Methods of treating or preventing respiratory conditions

ABSTRACT

The present disclosure provides methods of treating or preventing respiratory condition and/or for treating an IgE-mediated allergy and/or for reducing an allergic response to an allergen and/or for inducing anergy to an allergen in a subject and/or improving lung function in a subject suffering from an allergy comprising administering to a subject a population of cells enriched for STRO-1+ cells and/or progeny thereof and/or soluble factors derived therefrom.

RELATED APPLICATION DATA

The present application claims priority from U.S. Patent Application No.61/736352 entitled “Methods of treating or preventing respiratoryconditions” filed on 12 Dec. 2012. The entire contents of thatapplication are hereby incorporated by reference.

FIELD

The present disclosure relates to methods for treating or preventingrespiratory conditions, e.g., IgE-mediated allergic respiratoryconditions.

INTRODUCTION

Respiratory conditions are recognized as encompassing pathologicalconditions affecting the organs and tissues involved in gas exchange,and includes conditions of the upper respiratory tract, trachea,bronchi, bronchioles, alveoli, pleura and pleural cavity, and the nervesand muscles of breathing. Chronic respiratory conditions causeapproximately 7% of all deaths worldwide and represent about 4% of theglobal burden of disease. In the US alone, the cost of chronicrespiratory conditions is estimated to be about US$154 billion annually,including direct and indirect costs. Respiratory conditions can bedivided into several classes, including:

-   -   Inflammatory lung conditions, such as, asthma, cystic fibrosis,        emphysema, chronic obstructive pulmonary disorder or acute        respiratory distress syndrome, which are characterized by        increased levels of neutrophils and/or inflammatory cytokines in        the lungs of a subject;    -   obstructive lung conditions, such as chronic obstructive lung        disease and asthma, which are characterized by a reduction in        airway volume or impediment of free gas flow; and    -   restrictive lung conditions (also known as interstitial lung        diseases), such as infant respiratory distress syndrome, which        as characterized by loss of lung compliance causing incomplete        lung expansion and/or increased lung stiffness.

Asthma is a common chronic respiratory condition characterized byvariable and recurring symptoms, reversible airway obstruction, airway(e.g., bronchial) hyperresponsiveness, and an underlying inflammation.Acute symptoms of asthma include cough, wheezing, shortness of breathand nocturnal awakening. These symptoms usually arise from bronchospasmand require and respond to bronchodilator therapy. Central to thepathophysiology of asthma is the presence of underlying airwayinflammation mediated by the recruitment and activation of multiple celltypes including mast cells, eosinophils, T lymphocytes, macrophages,dendritic cells and neutrophils. The mechanisms influencing airwayhyperresponsiveness are multiple and include inflammation, dysfunctionalneuroregulation, and airway remodeling. Airway remodeling involvesstructural changes including thickening of the sub-basement membrane,subepithelial fibrosis, airway smooth muscle hypertrophy andhyperplasia, blood vessel proliferation and dilation with consequentpermanent changes in the airway that increase airflow obstruction andthat is not prevented by or fully reversible by current therapies.

Current standard therapies for asthma are a combination ofcorticosteroids and β2-agonists (anti-inflammatory and bronchodilatordrugs). These drugs provide acceptable control of the condition for manyasthmatics. However, it is estimated that 5 to 10% of the asthmapatients have symptomatic condition despite treatment with thiscombination of corticosteroids and β2-agonists (Chanez et al, J AllergyClin Immunol 119:1337-1348 (2007)).

Chronic obstructive pulmonary disease (COPD) is the most common chroniclung condition associated with significant morbidity and mortality. Inthe United States, COPD is the fourth leading cause of death andaccounts for more than $30 billion in annual health care costs. Anestimated 16 million adults are affected by COPD, and each year about120,000 Americans die of the condition. COPD is defined as a chronicdisease characterized by airway/alveolar/systemic inflammation, withmeasured airflow obstruction (FEV1/FVC<70% and FEVi<80% predicted) thatis partially improved with bronchodilator therapy. The local andsystemic release of inflammatory mediators by the lung cells leads toairway disease (chronic obstructive bronchitis) and, in a minority ofpatients, to destruction of parenchymal tissue (emphysema), both ofwhich can result in the airflow limitation that characterizes COPD. Therelease of these inflammatory mediators by the lung cells may alsoexacerbate inflammation in other organ systems, such as that observed incoronary, cerebrovascular, and peripheral vascular conditions.

Current therapies to treat COPD include bronchodilators, especiallyanticholinergic agents, that help to some degree decreasehyperinflation, therefore increasing inspiratory capacity and relievingdyspnea. Although corticosteroids are an effective treatment for mostcases of asthma, the inflammatory cells and mediators in COPD are notsensitive to treatment with systemic or inhaled corticosteroids makingtreatment with these agents of limited usefulness in COPD.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressives fibroticdisorder of the lower respiratory tract that typically affects adultsbeyond the age of 40. IPF is thought to occur as a result of initialinjury to the lung by environmental factors such as cigarette smokeleading to recruitment of neutrophils, lymphocytes and macrophages tothe lung alveoli. Release of fibrogenic cytokines, such as TGF-β byalveolar epithelial cells results in fibroblast proliferation,migration, and fibrosis. These fibroblasts not only fill the respiratoryspace but also secrete collagen and matrix proteins in response to manycytokines leading to parenchymal remodeling (Shimizu et al., Am J RespirCrit Care Med 163:210-217 (2001)). This differentiation of fibroblastsis likely key to the chronic nature of IPF. These events lead to coughand progressive shortness of breath. IPF patients have compromised lungfunction and have shown restrictive lung volumes and capacities.Although corticosteroids, immunosupressive agents, neutrophil elastaseinhibitor, hepatocyte growth factor, and interferon gamma-Ib have beenproposed as treatment agents for IPF, no treatment other than lungtransplantation is known to prolong survival and IPF remains a fataldisorder with a 3 to 6 yr median range of survival. Thus, the first lineof treatment of IPF has not yet been established.

Other respiratory conditions include, but are not limited to, pulmonaryarterial hypertension (PAH), pulmonary vasoconstriction,lymphangioleiomyomatosis (LAM), tuberous sclerosis complex (TSC), AcuteRespiratory Distress Syndrome (ARDS) and Ventilator Induced Lung Injury(VILI).

It will be apparent to the skilled artisan from the foregoing disclosurethat respiratory conditions are a prevalent and debilitating class ofconditions for which there are limited options for treatment. Thus, newtherapies for these conditions are desirable.

SUMMARY

The present inventors have now shown that using STRO-1⁺ cellpreparations they are able to reduce T_(H)2 mediated allergic responses(e.g., reduce eosinophils and/or IL-4 levels and/or IgE levels), e.g.,an IgE-mediated allergic response as well as bronchialhyperresponsiveness in a dose dependent manner in an accepted animalmodel of a human respiratory condition, such as, asthma, e.g., allergicasthma. The inventors found that they could suppress either (or both) anearly allergic response and/or a late allergic response. This doseresponsiveness demonstrates that it is the STRO-1⁺ cell preparationsthat is providing a therapeutic benefit.

The STRO-1⁺ cell preparations additionally reduced eosinophil cellinfiltration in the airway lumen and bronchoalveolar lavage fluid andneutrophil numbers in bronchoalveolar lavage fluid, demonstrating theability of these preparations to suppress inflammation in the lung of asubject, e.g., subjects suffering from an inflammatory respiratorycondition, such as, asthma.

The STRO-1⁺ cell preparations additionally reduced allergen specific IgElevels in treated animals.

The inventors also observed that late phase asthmatic response, e.g.,caused by migration of neutrophils and basophils to the respiratorysystem was improved in subjects receiving STRO-1⁺ cell preparations.These observations indicate that STRO-1⁺ cell preparations are usefulfor reducing or preventing damage to the respiratory system, e.g.,inflammation and/or remodeling caused by neutrophils and basophils.

The findings by the inventors provide the basis for a method of treatingor preventing a respiratory condition in a subject, the methodcomprising administering to the subject a population of cells enrichedfor STRO-1⁺ cells and/or progeny thereof and/or soluble factors derivedtherefrom.

The present disclosure additionally provides a method of treating orpreventing a IgE-mediated allergy (or a T_(H)2-mediated allergy) in asubject, the method comprising administering to the subject a populationof cells enriched for STRO-1⁺ cells and/or progeny thereof and/orsoluble factors derived therefrom.

The present disclosure additionally provides a method for reducing anallergic response to an allergen and/or for inducing anergy to anallergen, the method comprising administering to the subject apopulation of cells enriched for STRO-1⁺ cells and/or progeny thereofand/or soluble factors derived therefrom.

The present disclosure additionally provides a method for treating orpreventing an allergic response to house dust mite allergen (HDM) orreducing an allergic response to HDM and/or for inducing anergy to HDM,the method comprising administering to the subject a population of cellsenriched for STRO-1⁺ cells and/or progeny thereof and/or soluble factorsderived therefrom.

The present disclosure additionally provides a method for improving lungfunction in a subject, the method comprising administering to thesubject a population of cells enriched for STRO-1⁺ cells and/or progenythereof and/or soluble factors derived therefrom, wherein the subjectsuffers from an allergy, an IgE-mediated allergy or an allergic responseto HDM.

In one example, the respiratory condition is associated with excessivecell proliferation, remodeling, inflammation, vasoconstriction,bronchoconstriction, airway hyperreactivity and/or edema. For example,the disclosure provides methods for treating or preventing conditionssuch as asthma, chronic obstructive pulmonary disease, pulmonaryarterial hypertension; acute respiratory distress syndrome, ventilatorinduced lung injury, cystic fibrosis, bronchiectasis,alpha-1-antitrypsin deficiency, rhinitis, rhino sinusitis, primaryciliary dyskinesia, pneumonia, bronchiolitis, interstitial lung diseaseincluding lymphangioleiomyomatosis, idiopathic pulmonary fibrosis,obliterative bronchiolitis or bronchiolitis obliterans, nonspecificinterstitial pneumonia, cryptogenic organizing pneumonia, acuteinterstitial pneumonia, respiratory bronchiolitis-associatedinterstitial lung disease, or pulmonary sarcoidosis.

In one example, a lung or condition is an acute lung injury. Forexample, the acute lung injury is one or more of physical trauma, achemical injury, e.g., a chemical burn, smoke inhalation, or exposure toa toxic substance. In another specific embodiment, said lung disease,disorder, or condition is an injury caused by a neoplastic orparaneoplastic disease.

In one example, the respiratory condition is chronic. In this regard, amethod of the disclosure can be used to treat an early stage or latestage or both stages of a chronic respiratory condition.

In one example, the respiratory condition is an inflammatory respiratorycondition, an obstructive respiratory condition or a restrictiverespiratory condition.

In one example, the respiratory condition or allergy is a reversibleairway obstruction.

In one example, the respiratory condition or allergy is an obstructiverespiratory condition, such as, COPD, asthma, obliterative broncholitisor cystic fibrosis. In one example, the respiratory condition is asthma.

In one example, the respiratory condition is a restrictive respiratorycondition, such as, a restrictive lung condition (e.g., extrinsicallergic alveolitis, fibrosing alveolitis, asbestosis or eosinophilicpneumonia) or a restrictive pleural condition (e.g., pleural effusion,pneumothorax or bronchiectasis).

In one example, the respiratory condition is not due to an infection orcancer.

In one example, the respiratory condition is an inflammatory condition.For example, the condition is associated with airway hyperreactivityand/or bronchial hyperreactivity and/or eosinophil cell infiltration inthe airway lumen and bronchoalveolar lavage fluid. In this regard, inone example a method of the disclosure comprises administering apopulation of cells enriched for STRO-1⁺ cells and/or progeny thereofand/or soluble factors derived therefrom such that airwayhyperreactivity and/or bronchial hyperreactivity and/or eosinophil cellinfiltration and/or neutrophil infiltration in the airway lumen and/orbronchoalveolar lavage fluid is reduced.

In one example, the condition is asthma, such as chronic asthma or acuteasthma or allergic asthma. For example, the condition is chronic asthmaor allergic asthma.

In one example, the condition is associated with remodeling of the lung,e.g., asthma or pulmonary fibrosis, such as, idiopathic pulmonaryfibrosis.

In one example, the asthma is severe asthma and/or refractory asthma.

In one example, the condition is steroid refractory asthma. For example,a subject suffering from asthma is refractory to treatment with asteroid, e.g., a corticosteroid, such as flunisolide, mometasonefuroate, triamcinolone, fluticasone, budesonide, beclomethasonedipropionate or a combination of any two or more of the foregoing.

In another example, the condition is long acting beta agonist (LABA)refractory asthma. For example, a subject suffering from asthma isrefractory to treatment with a long acting beta agonist such as, forexample, salmeterol, formoterol, bumbeterol or clenbuterol.

In another example, the condition is LABA and steroid refractory asthma.

In one example, the method reduces or prevents an early phase allergicor asthmatic response.

In another example, the method reduces or prevents a late phase allergicor asthmatic response.

In one example, the condition is a fibrotic condition. The fibroticdisease of the lung may be interstitial lung disease (diffuseparenchymal lung disease). In another example, the interstitial lungdisease is silicosis, asbestosis, berylliosis, systemic sclerosis,polymyositis, or dermatomyositis. In other examples, the interstitiallung disease is caused by an antibiotic, a chemotherapeutic drug, anantiarrhythmic drug, or an infection.

In a further example, the condition is idiopathic pulmonary fibrosis.

In one example, a method as described herein in any example comprisesadministering a population of cells enriched for STRO-1^(bright) cellsand/or progeny thereof and/or soluble factors derived therefrom.

In one example, a method as described herein in any example comprisesadministering a population of cells enriched for STRO-1⁺ and tissuenon-specific alkaline phosphate⁺ (TNAP)⁺ cells and/or progeny thereofand/or soluble factors derived therefrom.

In one example, a method as described herein in any example comprisesadministering a population of cells enriched for tissue non-specificalkaline phosphate⁺ (TNAP)⁺ cells and/or progeny thereof and/or solublefactors derived therefrom. As shown herein, such cells are STRO-1⁺,e.g., STRO-1^(bright). In one example, the cells are enriched forSTRO-3⁺ cells.

In one example, the population enriched for STRO-1⁺ cells and/or progenythereof and/or soluble factors derived therefrom are administeredsystemically.

For example, the population and/or progeny and/or soluble factors areadministered intravenously.

In another example, the population and/or progeny and/or soluble factorsare administered intranasally or by inhalation.

In one example, the population and/or the progeny and/or the solublefactors are administered a plurality of times. In this regard, thepresent inventors have shown that a population of cells as describedherein can provide a therapeutic benefit for up to four weeks or for atleast four weeks. Accordingly, in one example, the population and/or theprogeny and/or the soluble factors are administered once every three ormore weeks. For example, the population and/or the progeny and/or thesoluble factors are administered once every four or more weeks. Forexample, the population and/or the progeny and/or the soluble factorsare administered once every five or more weeks. For example, thepopulation and/or the progeny and/or the soluble factors areadministered once every ten or more weeks. For example, the populationand/or the progeny and/or the soluble factors are administered onceevery twelve or more weeks.

In one example, the method comprises monitoring the subject andadministering a further dose of the population and/or the progeny and/orthe soluble factors when one or more of the following occurs:

-   (i) a subject begins to persistently wheeze and/or cough and/or have    chest tightness and/or have difficulty breathing;-   (ii) a subject shows one or more of the following when assessed by    spirometer:    -   a) 20% difference on at least three days in a week for at least        two weeks;    -   b) ≥20% improvement of peak flow following treatment, for        example:        -   10 minutes of inhaled β-agonist (e.g., salbutamol);        -   six weeks of inhaled corticosteroid (e.g., beclometasone);        -   14 days of 30 mg prednisolone.    -   c) ≥20% decrease in peak flow following exposure to a trigger        (e.g., exercise);-   (iii) bronchoscopy showing abnormal cells and/or foreign substances    and/or blockages in the respiratory tract of a subject; or-   (iv) chest CT scan showing abnormalities of the blood vessels in the    lungs, accumulation of blood or fluid in the lungs, bronchiectasis,    pleural effusion or pneumonia.

In one example, a method described herein according to any examplecomprises administering a dose of the population and/or the progenyand/or the soluble factors sufficient to achieve one or more of thefollowing:

-   (i) improved bronchial hyperresponsiveness, e.g., as assessed using    a bronchial challenge test;-   (ii) improved airway hyperresponsiveness;-   (iii) reduced eosinophil infiltration of the lung or bronchoalveolar    lavage fluid;-   (iv) reduced neutrophil infiltration of the lung or bronchoalveolar    lavage fluid;-   (v) reduced late asthmatic response, e.g., as assessed by    spirometer;-   (vi) reduced early asthmatic response, e.g., as assessed by    spirometer; and/or-   (vii) reduced lung remodeling/fibrosis, e.g., as assessed by chest    CT scan.

In one example, the dose is sufficient to achieve at least two or threeor four of five or all of the foregoing.

In one example, a method described herein according to any examplecomprises administering between 1×10⁶ to 150×10⁶ STRO-1⁺ cells and/orprogeny thereof.

In one example, a method described herein according to any examplecomprises administering between 25×10⁶ to 150×10⁶ STRO-1⁺ cells and/orprogeny thereof.

For example, the method comprises administering about 25×10⁶ or 75×10⁶or 150×10⁶ STRO-1⁺ cells and/or progeny thereof.

In one example, a method described herein according to any examplecomprises administering between about 2.5×10⁴ cells to 4.5×10⁶ STRO-1⁺cells and/or progeny thereof per kg.

In one example, a method described herein according to any examplecomprises administering between about 4.5×10⁵ to 4.5×10⁶ STRO-1⁺ cellsand/or progeny thereof per kg. For example, the method comprisesadministering about 4.5×10⁵ or about 5.5×10⁶ or about 1.7×10⁶ or about1.9×10⁶ or about 3.5×10⁶ or about 4.5×10⁶ STRO-1⁺ cells and/or progenythereof per kg.

In one example, a method described herein according to any examplecomprises administering a whole body dose of STRO-1⁺ cells and/orprogeny thereof and/or soluble factors derived therefrom. For example,when the cells or soluble factors are administered a plurality of times,the whole body dose remains constant.

For example, the method comprises administering 150×10⁶ STRO-1⁺ cellsand/or progeny thereof in 10 mL to a subject, i.e., 1.5×10⁶ STRO-1⁺cells and/or progeny thereof per mL.

In one example, a method described herein according to any examplecomprises administering to a subject suffering from steroid refractoryasthma or LABA refractory asthma or steroid and LABA refractory asthma150×10⁶ STRO-1⁺ cells and/or progeny thereof, e.g., in 10 mL to asubject, i.e., 1.5×10⁶ STRO-1⁺ cells and/or progeny thereof per mL.

In one example, a method described herein according to any examplecomprises administering to a subject suffering from idiopathic pulmonaryfibrosis 150×10⁶ STRO-1⁺ cells and/or progeny thereof, e.g., in 10 mL toa subject, i.e., 1.5×10⁶ STRO-1⁺ cells and/or progeny thereof per mL.

In one example, the population and/or the progeny cells are autogeneicor allogeneic and/or the soluble factors can be derived from autogeneicor allogeneic cells. In one example, the population and/or the progenyare allogeneic and/or the soluble factors are from allogeneic cells.

In accordance with the above example, the method can additionallycomprise obtaining the population and/or progeny cells and/or solublefactors or can additionally comprise isolating the population and/orprogeny cells and/or soluble factors. In one example, the populationand/or progeny cells are based on expression of STRO-1 and/or TNAP.

In one example, the population and/or progeny cells and/or solublefactors are obtained from the subject being treated. In another example,the population and/or progeny cells and/or soluble factors are obtainedfrom a different subject of the same species.

In one example, the population enriched for STRO-1⁺ cells and/or progenycells have been culture expanded prior to administration and/or prior toobtaining the soluble factors.

In accordance with the above example, a method as described hereinaccording to any example can additionally comprise culturing thepopulation and/or progeny cells.

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered in the form of acomposition comprising said STRO-1⁺ cells and/or progeny cells thereofand/or soluble factors derived therefrom and a carrier and/or excipient.

In accordance with the above example, a method as described hereinaccording to any example can additionally comprise formulating thepopulation and/or progeny and/or soluble factors into a composition.

In one example, the subject is suffering from a respiratory condition oran exacerbation thereof (e.g., an asthma attack) at the time oftreatment. For example, the subject is in need of treatment.

In one example, the subject has a respiratory condition, however is notactively suffering from the respiratory condition or an exacerbationthereof (e.g., an asthma attack) at the time of treatment, i.e., themethod is a method of preventing the condition or an exacerbationthereof.

The present disclosure also provides a population of cells enriched forSTRO-1⁺ cells and/or progeny thereof and/or soluble factors derivedtherefrom for use in the treatment or prevention of a respiratorycondition.

The present disclosure also provides for use of a population of cellsenriched for STRO-1⁺ cells and/or progeny thereof and/or soluble factorsderived therefrom in the manufacture of a medicament for treating orpreventing a respiratory condition in a subject.

The present disclosure also provides a kit comprising a population ofcells enriched for STRO-1⁺ cells and/or progeny thereof and/or solublefactors derived therefrom packaged with instructions for use in a methoddescribed herein according to any example.

For example, the present disclosure provides a kit comprising acomposition comprising the population and/or the progeny and/or thesoluble factors packaged with product information indicating use of thecomposition in a method described herein according to any example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Co-expression of TNAP (STRO-3) and the Mesenchymal PrecursorCell Marker, STRO-1^(bright) by Adult Human bone marrow morphonuclearcells (BMMNC). Dual-color immunofluorescence and flow cytometry wasperformed by incubation of STRO-1 MACS-selected BMMNC and indirectlylabeled with a goat anti-murine IgM antibody coupled to FITC (x axis),and STRO-3 mAb (murine IgG1) indirectly labeled with a goat anti-murineIgG coupled to PE (y axis). The dot plot histogram represents 5×10⁴events collected as listmode data. The vertical and horizontal lineswere set to the reactivity levels of <1.0% mean fluorescence obtainedwith the isotype-matched control antibodies, 1B5 (IgG) and 1A6.12 (IgM)treated under the same conditions. The results demonstrate that a minorpopulation of STRO-1^(bright) cells co-expressed TNAP (upper rightquadrant) while the remaining STRO-1⁺ cells failed to react with theSTRO-3 mAb.

FIG. 2. Graphical representations showing representative flow cytometrichistograms produced using single cell suspensions of culture expandedbone marrow derived cynomolgus MPCs with positive cell surfaceexpression of the mesenchymal stem cell markers, STRO-1, STRO-4 andCD146 (solid) relative to the isotype (IgM, IgG2a and IgG1) negativecontrols (hashed) detected using goat anti-murine IgM or IgGconjugated-FITC secondary antibodies. Representative histograms alsoshow that cynomolgus MPCs lack cell surface expression for markers ofmonocyte/macrophage (CD14), haematopietic stem/progenitor cells (CD34)and mature leukocyte (CD45). Levels of greater than 1% fluorescencecompared to the isotype control signify positivity.

FIG. 3 is a diagrammatic representation of the timeline of the study toassess the safety and efficacy of MPCs in treating a sheep model ofasthma.

FIG. 4 is a series of graphical representations showing early-phaseasthmatic response (EAR) over the course of the study for saline and MPCtreatment groups. Summary EAR data are shown in (A) for the controlgroup, and the three treatment groups, 25 million, 75 million, and 150million oMPCs. The data represents the percentage change in resistancefrom baseline resistance readings taken after control saline aerosolizedchallenge to peak resistance readings taken over the first hour afterallergen challenge. The EAR readings were taken on three occasionsthroughout the trial: 2 weeks before oMPC/saline treatment(pretreatment); 1 week after oMPC/saline treatment (1 wk posttreatment); and 4 weeks after oMPC/saline treatments (4 wk posttreatment). The data in (B) and (C) show comparisons between the controland treatment groups for the percentage change in EAR from pretreatmentto 1 week, and 4 weeks, after treatments respectively. Data is presentedas Mean±SEM. N=11 for control group and 75 million oMPC group; N=10 for25 million and 150 million oMPC group. **p<0.01*p<0.05.

FIG. 5 is series of graphical representations showing late phaseasthmatic response (LAR) over the course of the study for saline and MPCtreatment groups. Summary LAR data are shown in (A) for the controlgroup, and the three treatment groups, 25 million, 75 million, and 150million oMPCs. The data represents the percentage change in resistancefrom baseline resistance readings taken before aerosolized allergenchallenge to resistance readings taken 6 hours after allergen challenge.The LAR readings were taken on three occasions throughout the trial: 2weeks before oMPC/saline treatment (pretreatment); 1 week afteroMPC/saline treatment (1 wk post treatment); and 4 weeks afteroMPC/saline treatments (4 wk post treatment). The data in (B) and (C)show comparisons between the control and treatment groups for thepercentage change in LAR from pretreatment to 1 week, and 4 weeks, aftertreatments respectively. Data is presented as Mean±SEM. N=11 for controlgroup and 75 million oMPC group; N=10 for 25 million and 150 millionoMPC group. **p<0.01*p<0.05

FIG. 6 is a series of graphical representations showing bronchialhyperresponsiveness (BHR) over the course of the study for saline andMPC treatment groups. Summary BHR data are shown in (A) for the controlgroup, and the three treatment groups, 25 million, 75 million, and 150million oMPCs. The BHR data on the y axis represents the mean number ofbreath units of carbachol required to induce a 100% change inresistance. The BHR readings were taken on three occasions throughoutthe trial: 2 weeks before oMPC/saline treatment (pretreatment); 1 weekafter oMPC/saline treatment (1 wk post treatment); and 4 weeks afteroMPC/saline treatments (4 wk post treatment). The data in (B) and (C)show comparisons between the control and treatment groups for thepercentage change in BHR from pretreatment to 1 week, and 4 weeks, aftertreatments respectively. Data in (D) shows BHR data comparisons betweenthe control group and pooled treatment groups. Data is presented asMean±SEM. N=11 for control group and 75 million oMPC group; N=10 for 25million and 150 million oMPC group. *p<0.05**p<0.01

FIG. 7 is a series of graphical representations showing eosinophils inbronchoalveolar (BAL) fluid over the course of the study for saline andMPC treatment groups. Data are presented as a summary of percentageeosinophils (A), change in percentage eosinophils from pre-treatment at1 week (C) and 4 weeks (D) post treatment, and control group compared topooled treatment groups (E). Eosinophils/mL are shown in (B). Data ispresented as Mean±SEM. N=11 for control group and 75 million oMPC group;N=10 for 25 million and 150 million oMPC group. *p<0.05, **p<0.01.

FIG. 8 is a series of graphical representations showing neutrophils inbronchoalveolar (BAL) fluid over the course of the study for saline andMPC treatment groups. Data is presented as a summary of percentageneutrophils (A), and neutrophils/mL (B). Data is presented as Mean±SEM.N=11 for control group and 75 million oMPC group; N=10 for 25 millionand 150 million oMPC group. *p<0.05, **p<0.01, ***p<0.005

FIG. 9 is a series of graphical representations showing macrophages inbronchoalveolar (BAL) fluid over the course of the study for saline andMPC treatment groups. Data is presented as a summary of percentagemacrophages (A), and macrophages/mL (B). Data is presented as Mean±SEM.N=11 for control group and 75 million oMPC group; N=10 for 25 millionand 150 million oMPC group.

FIG. 10 is a series of graphical representations showing lymphocytes inbronchoalveolar (BAL) fluid over the course of the study for saline andMPC treatment groups. Data are presented as a summary of percentagelymphocytes (A), and lymphocytes/mL (B). Data is presented as Mean±SEM.N=11 for control group and 75 million oMPC group; N=10 for 25 millionand 150 million oMPC group.

FIG. 11 is a series of graphical representations showing IgE levels insera of asthmatic sheep. ELISA data showing mean absorbance (Abs) levelsfor HDM-specific IgE in the sera of trial sheep. Data is presented asmean±SEM and show comparisons of HDM-IgE levels before and after oMPCtreatments (A), and the percentage change in IgE levels frompre-treatment at 1 week (B) and 4 weeks (C). Pretreatment, 1 wkpost-treatment, and 4 week post-treatment sera were taken from all sheepon trial days 51, 72 and 93 respectively. N=11 for control group and 75million oMPC group; N=10 for 25 million and 150 million oMPC groups.*p<0.05**p<0.01.

DETAILED DESCRIPTION General Techniques and Selected Definitions

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Each example described herein is to be applied mutatis mutandis to eachand every other example of the disclosure unless specifically statedotherwise.

Those skilled in the art will appreciate that the present disclosure andindividual examples thereof are susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the disclosure includes all such variations andmodifications. The disclosure also includes all of the steps, features,compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specificexamples of the disclosure included herein, which are intended for thepurpose of exemplification only. Functionally-equivalent products,compositions and methods are clearly within the scope of the disclosureand examples thereof, as described herein.

The present disclosure is performed without undue experimentation using,unless otherwise indicated, conventional techniques of molecularbiology, microbiology, virology, recombinant DNA technology, peptidesynthesis in solution, solid phase peptide synthesis, and immunology.Such procedures are described, for example, in Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, New York, Second Edition (1989), whole of Vols I,

II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N.Glover, ed., 1985), IRL Press, Oxford, whole of text; OligonucleotideSynthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press,Oxford, whole of text, and particularly the papers therein by Gait,ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al,pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D.Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press,Oxford, whole of text; Perbal, B., A Practical Guide to MolecularCloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “TheChemistry of Peptide Synthesis” In: Knowledge database of Access to

Virtual Laboratory website (Interactiva, Germany); Sakakibara, D.,Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys.Res. Commun. 73 336-342; Merrifield, R. B. (1963). J Am. Chem. Soc. 85,2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides(Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press,New York. 12. Wünsch, E., ed. (1974) Synthese von Peptiden inHouben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15,4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984)Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky,M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis,Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. PeptideProtein Res. 25, 449-474; Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); and Animal Cell Culture: Practical Approach, ThirdEdition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source. In the context of soluble factorsderived from STRO-1⁺ cells and/or progeny cells thereof, this term shallbe taken to mean one or more factors, e.g., proteins, peptides,carbohydrates, etc, produced during in vitro culturing of STRO-1⁺ cellsand/or progeny cells thereof.

The term “respiratory condition” shall be taken to include any diseaseor condition that reduces lung function in a subject and includes, forexample, asthma, chronic bronchitis, emphysema, cystic fibrosis,respiratory failure, pulmonary oedema, pulmonary embolism, pulmonaryhypertension (high blood pressure), pneumonia and tuberculosis (TB),lung cancer, stiffening and scarring of lungs (e.g., caused by caused bydrugs, poisons, infections, or radiation), lung disorders from unusualatmospheric pressure (e.g., caused by a mechanical ventilator). In oneexample, the respiratory condition is a chronic lung condition and/or alung condition associated with inflammation in the lung, e.g., the lungcondition is asthma COPD or cystic fibrosis or pulmonary fibrosis orbronchiolitis or alveolitis or vasculitis or sarcoidosis. In anotherexample, the condition is associated with remodeling or fibrosis of asubject's lungs, e.g., the condition is pulmonary fibrosis (e.g.,idiopathic pulmonary fibrosis) or asthma.

As used herein the term “asthma” will be understood to mean a diseasecharacterized by paroxysmal or persistent symptoms of dyspnea, chesttightness, wheezing, sputum production and cough, associated withvariable airflow limitation and airway hyperresponsiveness to endogenousor exogenous stimuli (Canadian Asthma Consensus Guidelines) and/or acondition characterized by airway hyperresponsiveness that leads torecurrent episodes of wheezing, breathlessness, chest tightness, andcoughing, particularly at night or in the early morning along withvariable airflow obstruction which is often reversible eitherspontaneously or with treatment (The Global Initiative for Asthma).

As used herein, the term “severe asthma” will be understood to mean wellcontrolled asthma symptoms on high to very high doses of inhaledcorticosteroids, with or without the use of oral corticosteroids; and“very severe asthma” will be understood to mean well or not wellcontrolled asthma symptoms despite very high dose of inhaled andingested corticosteroids and with or without requiring additionaltherapies. For these definitions, the daily high and very high doses ofinhaled corticosteroid (approximate equivalent doses) are defined asfollows: High dose is beclomethasone diproprionate, 1000 to 2000 μg;fluticasone, 500 to 1000 μg; and budesonide, 800 to 1600 μg and veryhigh dose is fluticasone, 1000 to 2000 μg and budesonide, 1600-3200 μg.

As used herein, the term “refractory asthma” includes patients with“fatal” or “near fatal” asthma as well as the asthma subgroupspreviously described as “severe asthma” and “steroid-dependent and/orresistant asthma,” “difficult to control asthma,” “poorly controlledasthma,” “brittle asthma,” or “irreversible asthma.” Refractory asthmacan be defined as per the American Thoracic Society guidelines when oneor both major criteria and two minor criteria, described as follows, arefulfilled. The major criteria are: In order to achieve control to alevel of mild-moderate persistent asthma: (1) Treatment with continuousor near continuous (≥50% of year) oral corticosteroids 2) Requirementfor treatment with high-dose inhaled corticosteroids. The minor criteriaare: (1) Requirement for daily treatment with a controller medication inaddition to inhaled corticosteroids e.g., LABA, theophylline orleukotriene antagonist (2) Asthma symptoms requiring short-actingβ-agonist use on a daily or near daily basis (3) Persistent airwayobstruction (FEV₁<80% predicted; diurnal peak expiratory flow (PEF)variability>20%) (4) One or more urgent care visits for asthma per year(5) Three or more oral steroid “bursts” per year (6) Promptdeterioration with ≤5% reduction in oral or inhaled corticosteroid dose(7) Near fatal asthma event in the past. For the purposes of definitionof refractory asthma, the drug (μg/d) and the dose (puffs/d) are asfollows: (a) Beclomethasone dipropionate>1,260>40 puffs (42μg/inhalation)>20 puffs (84 μg/inhalation); (b) Budesonide>1,200>6puffs; (c) Flunisolide>2,000>8 puffs; (d) Fluticasone propionate>880>8puffs (110 μg), >4 puffs (220 μg); (e) Triamcinolone acetonide>2,000>20puffs.

As used herein, the term “acute asthma” or “allergic asthma” refers toasthma triggered by allergens (e.g., dust mite feces or pollen)activating mast cells located beneath the mucosa of the lower airways ofrespiratory tract. Activation of mast cells triggers release of granulesthat stimulate the nasal epithelium to produce mucus and subsequentcontraction of smooth muscle within the airway. This contraction ofsmooth muscle constricts the airway, causing the characteristicasthmatic wheezing.

“Chronic asthma” is not caused by allergens, but rather a result of theinflammation obtained from acute asthma. The overall effects of acuteasthma causes chronic inflammation, which causes the mucosal epitheliumto become hypersensitive to environmental responses. So simpleenvironmental agents, such as smoke, can stimulate the hypersensitiveepithelium to produce large amounts of mucous and constrict.

As used herein, the term “idiopathic pulmonary fibrosis” shall beunderstood to mean a chronic, progressive form of lung disease ofunknown origin characterized by fibrosis of the supporting framework(interstitium) of the lungs. Common symptoms are progressive dyspnea(difficulty breathing), but also include dry cough, clubbing (adisfigurement of the fingers), and rales (a crackling sound in the lungsduring inhalation, heard with a stethoscope). The 2002 ATS/ERSMultidisciplinary Consensus Statement on the Idiopathic InterstitialPneumonias proposed the following criteria for establishing thediagnosis of IPF without a lung biopsy:

-   Major criteria (all 4 required):    -   Exclusion of other known causes of interstitial lung disease        (drugs, exposures, connective tissue diseases);    -   Abnormal pulmonary function tests with evidence of restriction        (reduced vital capacity) and impaired gas exchange (pO2,        p(A-a)O2, DLCO);    -   Bibasilar reticular abnormalities with minimal ground glass on        high-resolution CT scans; and    -   Transbronchial lung biopsy or bronchoalveolar lavage (BAL)        showing no features to support an alternative diagnosis.-   Minor criteria (3 of 4 required):    -   Age>50;    -   Insidious onset of otherwise unexplained exertional dyspnea;    -   Duration of illness>3 months; and    -   Bibasilar inspiratory crackles.

The term “exacerbation” shall be understood to mean an exaggeration of arespiratory symptoms of a respiratory condition, e.g., an asthma attack.

An “early phase allergic response” (or asthmatic response) typicallyoccurs within 2 hours, or one hour or 30 minutes or 10 minutes or 1minute following allergen exposure and is also commonly referred to asthe immediate allergic reaction or as a Type I allergic reaction. Thereaction is caused by the release of histamine and mast cell granuleproteins by a process called degranulation, as well as the production ofleukotrienes, prostaglandins and cytokines, by mast cells following thecross-linking of allergen specific IgE molecules bound to mast cellFcεRI receptors. These mediators affect nerve cells causing itching,smooth muscle cells causing contraction (leading to the airway narrowingseen in allergic asthma), goblet cells causing mucus production, andendothelial cells causing vasodilatation and edema.

A “late phase allergic response” (or asthmatic response) generallydevelops about 6-12 hours or 8-12 hours after allergen exposure and ismediated by, e.g., mast cells). The products of the early phase reactioninclude chemokines and molecules that act on endothelial cells and causethem to express Intercellular adhesion molecule (such as vascular celladhesion molecule and selectins), which together result in therecruitment and activation of leukocytes from the blood into the site ofthe allergic reaction. Typically, the infiltrating cells observed inallergic reactions contain a high proportion of lymphocytes, andespecially, of eosinophils. The recruited eosinophils will degranulatereleasing a number of cytotoxic molecules (including Major Basic Proteinand eosinophil peroxidase) as well as produce a number of cytokines suchas IL-5. The recruited T-cells are typically of the Th2 variety and thecytokines they produce lead to further recruitment of mast cells andeosinophils, and in plasma cell isotype switching to IgE which will bindto the mast cell FcεRI receptors and prime the individual for furtherallergic responses

As used herein, the term “effective amount” shall be taken to mean asufficient quantity of STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom to reduce one or more symptoms of arespiratory condition as described herein.

As used herein, the term “therapeutically effective amount” shall betaken to mean a sufficient quantity of STRO-1⁺ cells and/or progenycells thereof and/or soluble factors derived therefrom to treat arespiratory condition, i.e., such that the subject no longer satisfiesthe clinical criteria for a respiratory condition or an exacerbationthereof.

As used herein, the term “prophylactically effective amount” shall betaken to mean a sufficient quantity of STRO-1⁺ cells and/or progenycells thereof and/or soluble factors derived therefrom to prevent orinhibit or delay the onset of a respiratory condition or an exacerbationthereof or a relapse thereof.

As used herein, the term “whole body dose” will be understood to meanthat subjects are administered a specified dose of cells and/or solublefactors irrespective of their body weight or body surface area.

As used herein, the term “treat” or “treatment” or “treating” shall beunderstood to mean administering a therapeutically effective amount ofsoluble factors and/or cells and reducing or inhibiting symptom(s) of arespiratory condition such that the subject is no longer clinicallydiagnosed with the condition or an exacerbation thereof.

As used herein, the term “prevent” or “preventing” or “prevention” shallbe taken to mean administering a prophylactically effective amount ofsoluble factors and/or cells and stopping or hindering or delaying thedevelopment or progression of a respiratory condition or exacerbationthereof. Preventing a respiratory condition also encompassesadministering a prophylactically effective amount of soluble factorsand/or cells and preventing or reducing the frequency of exacerbationsof the condition.

As used herein, the term “soluble factors” shall be taken to mean anymolecule, e.g., protein, peptide, glycoprotein, glycopeptide,lipoprotein, lipopeptide, carbohydrate, etc. produced by STRO-1⁺ cellsand/or progeny thereof that are water soluble. Such soluble factors maybe intracellular and/or secreted by a cell. Such soluble factors may bea complex mixture (e.g., supernatant) and/or a fraction thereof and/ormay be a purified factor. In one example, soluble factors are or arecontained within supernatant. Accordingly, any example herein directedto administration of one or more soluble factors shall be taken to applymutatis mutandis to the administration of supernatant.

As used herein, the term “supernatant” refers to the non-cellularmaterial produced following the in vitro culturing of STRO-1+ cellsand/or progeny thereof in a suitable medium, for example, liquid medium.Typically, the supernatant is produced by culturing the cells in themedium under suitable conditions and time, followed by removing thecellular material by a process such as centrifugation. The supernatantmay or may not have been subjected to further purification steps beforeadministration. In one example, the supernatant comprises less than 10⁵,more such as, less than 10⁴, for example, less than 10³, e.g., no livecells.

As used herein, the term “normal or healthy individual” shall be takento mean a subject that does not suffer from a respiratory condition asassessed by any method known in the art and/or described herein. In oneexample, a “normal or healthy individual” does not suffer from any ofthe symptoms of a respiratory condition.

Allergens

In one example, the present disclosure provides a method for reducing orpreventing a response (e.g., an allergic response) to an allergen. Asused herein the term “allergen” shall be taken to mean a substance thatcomprises one or more antigens that are capable of inducing specific IgEformation (i.e., an allergic response). Following production of IgE, theIgE is bound to a Fc receptor on the surface of a mast cell or abasophil. Following subsequent exposure to the allergen, at least twoIgE antibodies binding to at least two epitopes in the allergen causescross-linking of the Fab' regions of the IgE molecules resulting in mastcell or basophil release of a variety of vasoactive amine, such as, forexample, histamine, thereby inducing allergic symptoms. The termallergen includes all types of allergen, for example a polypeptideallergen, a phospholipid allergen, a fatty acid or a carbohydrate.Examples of common allergens are set forth in Table 1.

TABLE 1 Common allergens isolated from organisms Allergen sourceSystematic name Former name(s) MW Asterales Ambrosia artemisiifolia(short ragweed) Amb a 1 antigen E 38 Amb a 2 antigen K 38 Amb a 3 Ra3 11Amb a 5 Ra5 5 Amb a 6 Ra6 10 Amb a 7 Ra7 12 Amb a ? 11 Ambrosia trifida(giant ragweed) Amb t 5 Ra5G 4.4 Artemisia vulgaris (mugwort) Art v 2 35Poales Cynodon dactylon (Bermuda grass) Cyn d 1 32 Dactylis glomerata(orchard grass) Dac g 1 AgDg1 32 Dac g 2 11 Dac g 5 31 Lolium perenne(rye grass) Lol p 1 Group I 27 Lol p 2 Group II 11 Lol p 3 Group III 11Lol p 5 31 Lol p 9 Lol p Ib 31/35 Phleum pratense (timothy grass) Phl p1 27 Phl p 5 Ag25 32 Poa pratensis (Kentucky blue grass) Poa p 1 Group I33 Poa p 5 31 Poa p 9 32/34 Sorghum halepense (Johnson grass) Sor h 1Fagales Alnus glutinosa (alder) Aln g 1 17 Betula verrucosa (birch) Betv 1 17 Bet v 2 profilin 15 Carpinus betulus (hornbeam) Car b 1 17Corylus avellana (hazel) Cor a 1 17 Quercus alba (white oak) Que a 1 17Pinales Cryptomeria japonica (sugi) Cry j 1 41-45 Cry j 2 Junipersabinoides (mountain cedar) Jun s 1 50 Juniper virginiana (eastern redcedar) Jun v 1 45-50 Oleales Olea europea (olive) Ole e 1 16Dermatophagoides pteronyssinus (mite) Der p 1 Antigen P1 25 Der p 2 14Der p 3 trypsin 28/30 Der p 4 amylase 60 Der p 5 14 Der p 6 chymotrypsin25 Der p 7 22-28 Dermatophagoides microceras (mite) Der m 1 25Dermatophagoides farinae (mite) Der f 1 25 Der f 2 14 Der f 3 30Lepidoglyphus destructor (storage mite) Lep d ? 15 Canis familiaris(dog) Can f 1 25 Can f 2 27 Felis domesticus (cat saliva) Fel d 1 cat-138 Mus musculus Mus m 1 MUP 19 Rattus norvegius Rat n 1 17 Aspergillusfumigatus Asp f 1 18 Asp f ? 90 Asp f ? 55 Candida albicans Cand a 40Alternaria alternata Alt a 1 28 Trichophyton tonsurans Tri t 1 30Blattaria germanica (cockroach) Bla g 2 20

In one example, the allergen is from an animal, e.g., a mammal, e.g., adog or a cat or a rat or a mouse.

In one example, the allergen is from a plant, e.g., plant pollen.

In one example, the allergen is from an insect, e.g., a mite.

In one example, the allergen is HDM.

STRO-1⁺ Cells or Progeny Cells, and Supernatant or One or More SolubleFactors Derived Therefrom

STRO-1⁺ cells are cells found in bone marrow, blood, deciduous teeth(e.g., exfoliated deciduous teeth), dental pulp cells, adipose tissue,skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hairfollicles, intestine, lung, lymph node, thymus, bone, ligament, tendon,skeletal muscle, dermis, and periosteum.

In one example, STRO-1⁺ cells are capable of differentiating into one ormore or two or more and/or three germ lines such as mesoderm and/orendoderm and/or ectoderm.

In one example, the STRO-1⁺ cells are multipotential cells which arecapable of differentiating into a large number of cell types including,but not limited to, adipose, osseous, cartilaginous, elastic, muscular,and fibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. STRO-1⁺multipotential cells are thus non-hematopoietic progenitor cells whichdivide to yield daughter cells that are either stem cells or areprecursor cells which in time will irreversibly differentiate to yield aphenotypic cell.

In one example, the STRO-1⁺ cells are enriched from a sample obtainedfrom a subject, e.g., a subject to be treated or a related subject or anunrelated subject (whether of the same species or different). The terms“enriched”, “enrichment” or variations thereof are used herein todescribe a population of cells in which the proportion of one particularcell type or the proportion of a number of particular cell types isincreased when compared with an untreated population of the cells (e.g.,cells in their native environment). In one example, a populationenriched for STRO-1⁺ cells comprises at least about 0.1% or 0.5% or 1%or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% STRO-1⁺cells . In this regard, the term “population of cells enriched forSTRO-1⁺ cells ” will be taken to provide explicit support for the term“population of cells comprising X % STRO1⁺ cells ”, wherein X % is apercentage as recited herein.

The STRO-1⁺ cells can, in some examples, form clonogenic colonies, e.g.CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70%or 90% or 95%) can have this activity.

In one example, the population of cells is enriched from a cellpreparation comprising STRO-1⁺ cells in a selectable form. In thisregard, the term “selectable form” will be understood to mean that thecells express a marker (e.g., a cell surface marker) permittingselection of the STRO-1⁺ cells . The marker can be STRO-1, but need notbe. For example, as described and/or exemplified herein, cells (e.g.,MPCs) expressing STRO-2 and/or STRO-3 (TNAP) and/or STRO-4 and/or VCAM-1and/or CD146 and/or 3G5 also express STRO-1 (and can beSTRO-1^(bright)). Accordingly, an indication that cells are STRO-1⁺ doesnot mean that the cells are selected by STRO-1 expression. In oneexample, the cells are selected based on at least STRO-3 expression,e.g., they are STRO-3⁺ (TNAP⁺).

Reference to selection of a cell or population thereof does not requireselection from a specific tissue source. As described herein STRO-1⁺cells can be selected from or isolated from or enriched from a largevariety of sources. That said, in some examples, these terms providesupport for selection from any tissue comprising STRO-1⁺ cells (e.g.,MPCs) or vascularized tissue or tissue comprising pericytes (e.g.,STRO1⁺ pericytes) or any one or more of the tissues recited herein.

In one example, the cells used in methods of the present disclosureexpress one or more markers individually or collectively selected fromthe group consisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, STRO-4⁺(HSP-90β), CD45⁺, CD146⁺, 3G5⁺ or any combination thereof.

By “individually” is meant that the disclosure encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the disclosure encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

For example, the STRO-1⁺ cells are STRO-1^(bright) (syn. STRO-1^(bri)).In one example, the Stro-1^(bri) cells are preferentially enrichedrelative to STRO-1^(dim) or STRO-1^(intermediate) cells.

In one example, the STRO-1^(bright) cells are additionally one or more(or all) of TNAP⁺, VCAM-1⁺, THY-1^(+,)STRO-2⁺, STRO-4⁺ (HSP-90β) and/orCD146⁺. For example, the cells are selected for one or more of theforegoing markers and/or shown to express one or more of the foregoingmarkers. In this regard, a cell shown to express a marker need not bespecifically tested, rather previously enriched or isolated cells can betested and subsequently used, isolated or enriched cells can bereasonably assumed to also express the same marker.

In one example, the mesenchymal precursor cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This term means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labeled or is undetectableabove background levels, e.g., levels detected suing an isotype controlantibody.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectably labeled.Whilst not wishing to be limited by theory, it is proposed that “bright”cells express more of the target marker protein (for example the antigenrecognized by STRO-1) than other cells in the sample. For instance,STRO-1^(bri) cells produce a greater fluorescent signal, when labeledwith a FITC-conjugated STRO-1 antibody as determined by fluorescenceactivated cell sorting (FACS) analysis, than non-bright cells(STRO-1^(dull/dim)). In one example, “bright” cells constitute at leastabout 0.1% of the most brightly labeled cells (e.g., bone marrowmononuclear cells) contained in the starting sample. In other examples,“bright” cells constitute at least about 0.1%, at least about 0.5%, atleast about 1%, at least about 1.5%, or at least about 2%, of the mostbrightly labeled cells, e.g., bone marrow mononuclear cells contained inthe starting sample. In one example, STRO-1^(bright)cells have 2 logmagnitude higher expression of STRO-1 surface expression relative to“background”, namely cells that are STRO-1″. By comparison, STRO-1^(dim)and/or STRO-1^(intermediate) cells have less than 2 log magnitude higherexpression of STRO-1 surface expression, typically about 1 log or lessthan “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In one example, the TNAP is BAP. In one example,TNAP as used herein refers to a molecule which can bind the STRO-3antibody produced by the hybridoma cell line deposited with ATCC on 19Dec. 2005 under the provisions of the Budapest Treaty under depositaccession number PTA-7282.

Furthermore, in a preferred example, the STRO-1⁺ cells are capable ofgiving rise to clonogenic CFU-F.

In one example, a significant proportion of the STRO-1⁺ multipotentialcells are capable of differentiation into at least two different germlines. Non-limiting examples of the lineages to which the multipotentialcells may be committed include bone precursor cells; hepatocyteprogenitors, which are multipotent for bile duct epithelial cells andhepatocytes; neural restricted cells, which can generate glial cellprecursors that progress to oligodendrocytes and astrocytes; neuronalprecursors that progress to neurons; precursors for cardiac muscle andcardiomyocytes, glucose-responsive insulin secreting pancreatic betacell lines. Other lineages include, but are not limited to,odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another example, the STRO-1⁺ cells are not capable of giving rise,upon culturing, to hematopoietic cells.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used. In another useful example of the disclosure, cells of anon-human animal (or if the patient is not a human, from anotherspecies) are used.

The present disclosure also contemplates use of supernatant or solublefactors obtained or derived from STRO-1⁺ cells and/or progeny cellsthereof (the latter also being referred to as expanded cells) which areproduced from in vitro culture. Expanded cells of the disclosure may ahave a wide variety of phenotypes depending on the culture conditions(including the number and/or type of stimulatory factors in the culturemedium), the number of passages and the like. In certain examples, theprogeny cells are obtained after about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, or about 10 passages from theparental population. However, the progeny cells may be obtained afterany number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an example, progeny cells useful for the methods of the disclosureare obtained by isolating TNAP⁺ STRO-1⁺ cells from bone marrow usingmagnetic beads labeled with the STRO-3 antibody, and then cultureexpanding the isolated cells (see Gronthos et al. Blood 85: 929-940,1995 for an example of suitable culturing conditions).

In one example, such expanded cells (progeny) (for example, after atleast 5 passages) can be TNAP⁻, CC9⁺, HLA class I⁺, HLA class II³¹ ,CD14⁻, CD19⁻, CD3⁻, CD11a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one example, expanded cells stillhave the capacity to differentiate into different cell types.

In one example, an expended cell population used to obtain supernatantor soluble factors, or cells per se, comprises cells wherein at least25%, e.g., at least 50%, of the cells are CC9⁺.

In another example, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, e.g., at least 45%, of the cells are STRO-1⁺.

In a further example, the expanded cells may express one or more markerscollectively or individually selected from the group consisting ofLFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-4(HSP-90β), STRO-1^(bright) and CD146 or any combination of thesemarkers.

In one example, the progeny cells are Multipotential Expanded STRO-1⁺Multipotential cells Progeny (MEMPs) as defined and/or described in WO2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising MPCs from which progeny are derived maybe directly harvested from a tissue source, or alternatively it may be apopulation that has already been expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP, STRO-4 (HSP-90β), SIRO-1^(bright),3G5⁺, VCAM-1, THY-1, CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested STRO-1⁺ multipotentialcells in that they are positive for the marker STRO-1⁺ and negative forthe marker Alkaline phosphatase (ALP). In contrast, freshly isolatedSTRO-1⁺ multipotential cells are positive for both STRO-1^(bri) andALP⁻. In one example of the present disclosure, at least 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have thephenotype STRO-1^(bri), ALP⁻. In a further example the MEMPS arepositive for one or more of the markers Ki67, CD44 and/or CD49c/CD29,VLA-3, α3β1. In yet a further example the MEMPs do not exhibit TERTactivity and/or are negative for the marker CD18.

The STRO-1⁺ cell starting population may be derived from any one or moretissue types set out in WO 01/04268 or WO 2004/085630, namely bonemarrow, dental pulp cells, adipose tissue and skin, or perhaps morebroadly from adipose tissue, teeth, dental pulp, skin, liver, kidney,heart, retina, brain, hair follicles, intestine, lung, spleen, lymphnode, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletalmuscle.

It will be understood that in performing methods described in thepresent disclosure, separation of cells carrying any given cell surfacemarker can be effected by a number of different methods, however,exemplary methods rely upon binding a binding agent (e.g., an antibodyor antigen binding fragment thereof) to the marker concerned followed bya separation of those that exhibit binding, being either high levelbinding, or low level binding or no binding. The most convenient bindingagents are antibodies or antibody-based molecules, for examplemonoclonal antibodies or based on monoclonal antibodies (e.g., proteinscomprising antigen binding fragments thereof) because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. For example, the separation techniques maximizethe retention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques.

In one example, the method for isolating STRO-1⁺ cells comprises a firststep being a solid phase sorting step utilizing for example magneticactivated cell sorting (MACS) recognizing high level expression ofSTRO-1. A second sorting step can then follow, should that be desired,to result in a higher level of precursor cell expression as described inpatent specification WO 01/14268. This second sorting step might involvethe use of two or more markers.

The method obtaining STRO-1⁺ cells might also include the harvesting ofa source of the cells before the first enrichment step using knowntechniques. Thus the tissue will be surgically removed. Cells comprisingthe source tissue will then be separated into a so called single cellssuspension. This separation may be achieved by physical and or enzymaticmeans.

Once a suitable STRO-1⁺ cell population has been obtained, it may becultured or expanded by any suitable means to obtain MEMPs.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used to obtain the supernatant or soluble factors. In anotheruseful example of the disclosure, cells of a non-human animal (or if thepatient is not a human, from another species) are used to obtainsupernatant or soluble factors.

Methods and uses of the present disclosure can be practiced using cellsfrom any non-human animal species, including but not limited tonon-human primate cells, ungulate, canine, feline, lagomorph, rodent,avian, and fish cells. Primate cells with which methods of thedisclosure may be performed include but are not limited to cells ofchimpanzees, baboons, cynomolgus monkeys, and any other New or Old Worldmonkeys. Ungulate cells with which the disclosure may be performedinclude but are not limited to cells of bovines, porcines, ovines,caprines, equines, buffalo and bison. Rodent cells with which thedisclosure may be performed include but are not limited to mouse, rat,guinea pig, hamster and gerbil cells. Examples of lagomorph species withwhich the disclosure may be performed include domesticated rabbits, jackrabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which methods ofthe disclosure may be performed.

In one example, the cells are human cells.

Cells useful for the methods of the disclosure may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Anymethod maintaining the biological activity of the isolated stem cellssuch as mesenchymal stem/progenitor cells, or progeny thereof, may beutilized in connection with the present disclosure. In one example, thecells are maintained and stored by using cryo-preservation.

Genetically-Modified Cells

In one example, the STRO-1⁺ cells and/or progeny cells thereof aregenetically modified, e.g., to express and/or secrete a protein ofinterest. For example, the cells are engineered to express a proteinuseful in the treatment of a respiratory condition, such as, a protease,a DNAse or a surfactant protein, e.g., surfactant protein C.

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present example of the disclosure.

In one example, the nucleic acid is provided in the form of anexpression construct. As used herein, the term “expression construct”refers to a nucleic acid that has the ability to confer expression on anucleic acid (e.g. a reporter gene and/or a counter-selectable reportergene) to which it is operably connected, in a cell. Within the contextof the present disclosure, it is to be understood that an expressionconstruct may comprise or be a plasmid, bacteriophage, phagemid, cosmid,virus sub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the disclosure will be apparent to the skilled artisanand are described, for example, in Ausubel et al (In: Current Protocolsin Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable formethods of the present disclosure in a mammalian cell is, for example, avector of the pcDNA vector suite supplied by Invitrogen, a vector of thepCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Life Technologies Corporation,Clontech or Promega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the disclosure is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include, for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739(1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al,Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller andRosman BioTechniques 7:980-990, 1989; Miller, A. D. Human Gene Therapy7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc.Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 andWO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka.Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, HumanGene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169,1994; and Zhou et al. J Exp. Med. 179:1867-1875, 1994.

Additional viral vectors useful for delivering an expression constructof the disclosure include, for example, those derived from the poxfamily of viruses, such as vaccinia virus and avian poxvirus or analphavirus or a conjugate virus vector (e.g. that described inFisher-Hoch et al., Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Assaying Therapeutic/Prophylactic Potential of Cells and Soluble Factors

Methods for determining the ability of cells or soluble factors to treator prevent or delay the onset or progression of a respiratory conditionwill be apparent to the skilled artisan.

For example, cells or soluble factors (e.g., a mixture of factors or asingle factor or a fraction of factors (e.g., derived by affinitypurification or chromatography)) are administered to a model of arespiratory condition and the effect on one or more symptoms isassessed.

Exemplary models of respiratory conditions include an animal model ofallergy, e.g., allergic asthma, such as a model described inWO2002/098216, a mouse model of allergic asthma, e.g., induced by hostdust mite protein (Fattouh et al., Am J Respir Crit Care Med 172:314-321, 2005), a mouse model of severe asthma in which IL-5 and eotaxinare overexpressed, mice receiving intratracheal instillation ofpoly-1-lysine which are hypersensitive to methacholine when delivered asan aerosol (Homma et al., Am J Physiol Lung Cell Mol Physiol 289:L413-L418, 2005), bleomycin or FITC or silica induced models ofpulmonary fibrosis (Muggia et al., Cancer Treat Rev 10: 221-243, 1983;Roberts et al., J Pathol 176: 309-318, 1995; Oberdorster Inhal Toxicol8: 73-89, 1996).

It will be apparent to the skilled artisan from the foregoing that thepresent disclosure also provides a method for identifying or isolating acell or a soluble factor for the treatment, prevention or delay of arespiratory condition, the method comprising:

-   -   (i) administering a cell or a soluble factor to a test subject        suffering from a respiratory condition and assessing a symptom        of the respiratory condition;    -   (ii) comparing the symptom of respiratory condition levels of        the subject at (i) to the symptom of respiratory condition of a        control subject suffering from the respiratory condition to        which the cell or soluble factor has not been administered,        wherein an improvement in the symptom in the test subject        compared to the control subject indicates that the cell or        soluble factor treats respiratory condition.

The cell may be any cell described herein according to any example.

Exemplary symptoms are described herein.

Cellular Compositions

In one example of the present disclosure STRO-1⁺ cells and/or progenycells thereof are administered in the form of a composition. In oneexample, such a composition comprises a pharmaceutically acceptablecarrier and/or excipient.

The terms “carrier” and “excipient” refer to compositions of matter thatare conventionally used in the art to facilitate the storage,administration, and/or the biological activity of an active compound(see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., MacPublishing Company (1980). A carrier may also reduce any undesirableside effects of the active compound. A suitable carrier is, for example,stable, e.g., incapable of reacting with other ingredients in thecarrier. In one example, the carrier does not produce significant localor systemic adverse effect in recipients at the dosages andconcentrations employed for treatment.

Suitable carriers for the present disclosure include thoseconventionally used, e.g., water, saline, aqueous dextrose, lactose,Ringer's solution, a buffered solution, hyaluronan and glycols areexemplary liquid carriers, particularly (when isotonic) for solutions.Suitable pharmaceutical carriers and excipients include starch,cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium stearate, sodium stearate, glycerol monostearate,sodium chloride, glycerol, propylene glycol, water, ethanol, and thelike.

In another example, a carrier is a media composition, e.g., in which acell is grown or suspended. For example, such a media composition doesnot induce any adverse effects in a subject to whom it is administered.

Exemplary carriers and excipients do not adversely affect the viabilityof a cell and/or the ability of a cell to reduce, prevent or delay arespiratory condition.

In one example, the carrier or excipient provides a buffering activityto maintain the cells and/or soluble factors at a suitable pH to therebyexert a biological activity, e.g., the carrier or excipient is phosphatebuffered saline (PBS). PBS represents an attractive carrier or excipientbecause it interacts with cells and factors minimally and permits rapidrelease of the cells and factors, in such a case, the composition of thedisclosure may be produced as a liquid for direct application to theblood stream or into a tissue or a region surrounding or adjacent to atissue, e.g., by injection.

STRO-1⁺ cells and/or progeny cells thereof can also be incorporated orembedded within scaffolds that are recipient-compatible and whichdegrade into products that are not harmful to the recipient. Thesescaffolds provide support and protection for cells that are to betransplanted into the recipient subjects. Natural and/or syntheticbiodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in thepractice of methods of the disclosure. Exemplary scaffolds include, butare not limited to biological, degradable scaffolds. Naturalbiodegradable scaffolds include collagen, fibronectin, and lamininscaffolds. Suitable synthetic material for a cell transplantationscaffold should be able to support extensive cell growth and cellfunction. Such scaffolds may also be resorbable. Suitable scaffoldsinclude polyglycolic acid scaffolds, e.g., as described by Vacanti, etal. J. Ped. Surg. 23:3-9 1988; Cima, et al. Biotechnol. Bioeng. 38:1451991; Vacanti, et al. Plast. Reconstr. Surg. 88:753-9 1991; or syntheticpolymers such as polyanhydrides, polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold(such as Gelfoam from Upjohn Company.

The cells may be administered as a component of a pharmaceuticalcomposition specifically formulated for intranasal administration. Incertain examples, the cells are co-administered with an enzyme inhibitoror an absorption enhancer. In other examples, the pharmaceuticalcompositions formulated for intranasal administration comprise enzymeinhibitor and/or absorption enhancers. In yet other examples, thepharmaceutical compositions comprise synthetic surfactants, bile salts,phospholipids, and cylodextrins. The cells may also be intranasallyadministered via an emulsion or a liposome. In certain examples,intranasal administration is achieved by use of polymeric microspheres.The cells may be administered in the presence of sodium glycohcholate(NaGC) and linoleic acid.

The pharmaceutical composition for intranasal administration may beadministered as a spray, aerosol, gel, solution, emulsion, orsuspension. Alternatively, the pharmaceutical composition isadministered directly to the upper airways such as e.g. the paranasalsinuses. In one example, the cells or the pharmaceutical composition areadministered via a microcatheter.

The cellular compositions useful for methods described herein may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentdisclosure include, but are not limited to, other multipotent orpluripotent cells or stem cells, or bone marrow cells. The cells ofdifferent types may be admixed with a composition of the disclosureimmediately or shortly prior to administration, or they may beco-cultured together for a period of time prior to administration.

In one example, the composition comprises an effective amount or atherapeutically or prophylactically effective amount of cells. Exemplarydosages are described herein. The exact amount of cells to beadministered is dependent upon a variety of factors, including the age,weight, and sex of the patient, and the extent and severity of therespiratory condition.

In some examples, cells are contained within a chamber that does notpermit the cells to exit into a subject's circulation, however thatpermits factors secreted by the cells to enter the circulation. In thismanner soluble factors may be administered to a subject by permittingthe cells to secrete the factors into the subject's circulation. Such achamber may equally be implanted at a site in a subject to increaselocal levels of the soluble factors, e.g., implanted in or near apancreas.

In some examples of the disclosure, it may not be necessary or desirableto immunosuppress a patient prior to initiation of therapy with cellularcompositions. Accordingly, transplantation with allogeneic, or evenxenogeneic, STRO-1⁺ cells or progeny thereof may be tolerated in someinstances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy and/or reduce an immune response of a subject against thecellular composition. This may be accomplished through the use ofsystemic or local immunosuppressive agents, or it may be accomplished bydelivering the cells in an encapsulated device. The cells may beencapsulated in a capsule that is permeable to nutrients and oxygenrequired by the cell and therapeutic factors the cell is yet impermeableto immune humoral factors and cells. For example, the encapsulant ishypoallergenic, is easily and stably situated in a target tissue, andprovides added protection to the implanted structure. These and othermeans for reducing or eliminating an immune response to the transplantedcells are known in the art. As an alternative, the cells may begenetically modified to reduce their immunogenicity.

Compositions of Soluble Factors

In one example, STRO-1⁺ cell-derived and/or progeny cell-derivedsupernatant or soluble factors are administered in the form of acomposition, e.g., comprising a suitable carrier and/or excipient. Inone example, the carrier or excipient does not adversely affect thebiological effect of the soluble factors or supernatant.

In one example, the composition comprises a composition of matter tostabilize a soluble factor or a component of supernatant, e.g., aprotease inhibitor. In one example, the protease inhibitor is notincluded in an amount sufficient to have an adverse effect on a subject.

Compositions comprising STRO-1⁺ cell-derived and/or progeny cell-derivedsupernatant or soluble factors may be prepared as appropriate liquidsuspensions, e.g., in culture medium or in a stable carrier or a buffersolution, e.g., phosphate buffered saline. Suitable carriers aredescribed herein above. In another example, suspensions comprisingSTRO-1⁺ cell-derived and/or progeny cell-derived supernatant or solublefactors are oily suspensions for injection. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil; or synthetic fattyacid esters, such as ethyl oleate or triglycerides; or liposomes.Suspensions to be used for injection may also contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Sterile injectable solutions can be prepared by incorporating thesupernatant or soluble factors in the required amount in an appropriatesolvent with one or a combination of ingredients described above, asrequired, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the supernatant orsoluble factors into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, exemplary methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In accordance with an alternative example of thedisclosure, the supernatant or soluble factors may be formulated withone or more additional compounds that enhance its solubility.

Other exemplary carriers or excipients are described, for example, inHardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In some cases, isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride are includedin the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, monostearate salts and gelatin.Moreover, the soluble factors may be administered in a time releaseformulation, for example in a composition which includes a slow releasepolymer. The active compounds can be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art.

The supernatant or soluble factors may be administered in combinationwith an appropriate matrix, for instance, to provide slow release of thesoluble factors.

Additional Components of Compositions

The STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof may be administered with other beneficial drugs orbiological molecules (growth factors, trophic factors). Whenadministered with other agents, they may be administered together in asingle pharmaceutical composition, or in separate pharmaceuticalcompositions, simultaneously or sequentially with the other agents(either before or after administration of the other agents). Bioactivefactors which may be co-administered include anti-apoptotic agents(e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspaseinhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors,TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,TRANILAST, REIVIICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPDXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine) as well as local anesthetics.

In one example, a composition as described herein according to anyexample comprises an anti-inflammatory agent, an immunomodulatory agent,an immunosuppressive agent, a pain medication, or an antibiotic. In oneexample, the second therapeutic agent is an immunomodulatory agent. Inanother example, the second agent is an anti-CD3 antibody (e.g., OKT3,muronomab), an anti-IL-2 receptor antibody (e.g., basiliximab anddaclizumab), an anti T cell receptor antibody (e.g., Muromonab-CD3),azathioprine, a calcineurin inhibitor, a corticosteroid, cyclosporine,methotrexate, mercaptopurine, mycophenolate mofetil, tacrolimus, orsirolimus.

Alternatively, or in addition, cells, secreted factors and/or acomposition as described herein according to any example is combinedwith a known treatment of a respiratory condition, e.g., a steroid orLABA.

In one example, a pharmaceutical composition as described hereinaccording to any example comprises a compound used to treat arespiratory condition. Alternatively, a method of treatment/prophylaxisas described herein according to any example of the disclosureadditionally comprises administering a compound used to treatrespiratory condition. Exemplary compounds are described herein and areto be taken to apply mutatis mutandis to these examples of the presentdisclosure.

In another example, a composition as described herein according to anyexample additionally comprises a factor that induces or enhancesdifferentiation of a progenitor cell into a vascular cell. Exemplaryfactors include, vascular endothelial growth factor (VEGF), plateletderived growth factor (PDGF; e.g., PDGF-BB), and FGF.

In another example, a composition as described herein according to anyexample additionally comprises a tissue specific committed cell (TSCC).In this respect, International Patent Application No. PCT/AU2005/001445demonstrates that administration of a TSCC and a STRO-1⁺ cells can leadto enhanced proliferation of the TSCC. In one example, the TSCC is avascular cell. Administration of such a composition to a subject maylead to increased production of vasculature, e.g., leading to increasednutrients being delivered to the affected tissue.

Medical Devices

The present disclosure also provides medical devices for use or whenused in a method as described herein according to any example. Forexample, the present disclosure provides a syringe or catheter orinhaler or other suitable delivery device comprising STRO-1⁺ cellsand/or progeny cells thereof and/or soluble factors therefrom and/or acomposition as described herein according to any example. Optionally,the syringe or catheter or inhaler is packaged with instructions for usein a method as described herein according to any example.

In another example, the present disclosure provides an implantcomprising STRO-1⁺ cells and/or progeny cells thereof and/or solublefactors therefrom and/or a composition as described herein according toany example. Optionally, the implant is packaged with instructions foruse in a method as described herein according to any example. Suitableimplants may be formed with a scaffold, e.g., as described herein aboveand STRO-1⁺ cells and/or progeny cells thereof and/or soluble factorstherefrom.

Modes of Administration

The STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof may be surgically implanted, injected, inhaled,delivered (e.g., by way of a catheter or syringe), or otherwiseadministered directly or indirectly to the site in need of repair oraugmentation, e.g., into a lung.

In on example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof is/are delivered to the blood stream ofa subject. For example, the STRO-1⁺ cell-derived supernatant or solublefactors, STRO-1⁺ cells or progeny thereof are delivered parenterally.Exemplary routes of parenteral administration include, but are notlimited to, intraperitoneal, intraventricular, intracerebroventricular,intrathecal, or intravenous. In one example, the STRO-1⁺ cell-derivedsupernatant or soluble factors, STRO-1⁺ cells or progeny thereof aredelivered intra-arterially, into an aorta, into an atrium or ventricleof the heart or into a blood vessel, e.g., intravenously. In thisregard, STRO-1⁺ cells have been shown to migrate to sites of injuryand/or to the lungs.

In the case of cell delivery to an atrium or ventricle of the heart,cells can be administered to the left atrium or ventricle to avoidcomplications that may arise from rapid delivery of cells to the lungs.

In one example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are delivered intravenously.

In one example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are injected into the site of delivery,e.g., using a syringe or through a catheter or a central line.

In one example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are delivered intranasally or byinhalation.

Selecting an administration regimen for a therapeutic formulationdepends on several factors, including the serum or tissue turnover rateof the entity, the level of symptoms, and the immunogenicity of theentity. In one example, an administration regimen maximizes the amountof cells and/or factors delivered to the subject consistent with anacceptable level of side effects. Accordingly, the amount of cellsand/or factors delivered depends in part on the particular entity andthe severity of the condition being treated.

In one example, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are delivered as a single bolus dose.Alternatively, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are administered by continuousinfusion, or by doses at intervals of, e.g., one day, one week, or 1-7times per week. An exemplary dose protocol is one involving the maximaldose or dose frequency that avoids significant undesirable side effects.A total weekly dose depends on the type and activity of thecompound/cell being used. Determination of the appropriate dose is madeby a clinician, e.g., using parameters or factors known or suspected inthe art to affect treatment or predicted to affect treatment. Generally,the dose begins with an amount somewhat less than the optimum dose andis increased by small increments thereafter until the desired or optimumeffect is achieved relative to any negative side effects.

The present inventors have shown therapeutic benefits provided bySTRO-1⁺ cells and/or progeny thereof and/or soluble factors derivedtherefrom are observed for at least four weeks in a subject.Accordingly, in some examples the cells are administered weekly,fortnightly, once every three weeks or once every four weeks.

In accordance with examples of the disclosure directed to treating ordelaying the progression of a respiratory condition, STRO-1⁺ cellsand/or progeny cells thereof and/or soluble factors derived therefromare administered following diagnosis of the disorder, e.g., usingstandard methods known in the art and/or described herein.

For those examples directed to preventing or delaying the onset ofrespiratory condition, the STRO-1⁺ cells and/or progeny cells thereofand/or soluble factors derived therefrom can administered prior toclinical diagnosis of the disorder.

In one example, a method of treatment of the disclosure comprisesassessing a treated subject for improvement in one or more parameters oflung function after administration (e.g., from 7 days to 30 daysafterwards), wherein the parameters of lung function are forcedexpiratory volume in one second (FEV₁); forced volume vital capacity(FVC); FEV₁/FVC; peak expiratory flow (PEF); forced expiratory flow25%-50% or 25% 75% (average flow of air exiting the lung during themiddle portion of the expiration); forced expiratory time (FET); totallung capacity (TLC); diffusing capacity, carbon monoxide (DLCO); maximumvoluntary ventilation; a detectable improvement in one or more of achest X-ray, CT scan, MRI, bronchoscopy or similar scan (e.g., visibleimprovement in the appearance of the lung); or a detectable improvementin the level of carbon dioxide detectable in the blood (e.g., movementof CO₂ levels to within a normal range). In one example, theadministration results in improvement of one or more of the parametersof lung function (1) to 80% or more of expected; or (2) by at least 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 50%. In one example, the method comprisesidentifying any of the parameters that, prior to administration, areless than 80% of expected values for an individual of the same heightand weight, and assessing said parameters after treatment, whereintreatment results in improvement of one or more of said parameters oflung function (1) to 80% or more of expected; or (2) by at least 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 50%.

The present disclosure includes the following non-limiting examples.

EXAMPLES Example 1: Immunoselection of MPCs by Selection of STRO-3⁺Cells

Bone marrow (BM) is harvested from healthy normal adult volunteers(20-35 years old). Briefly, 40 ml of BM is aspirated from the posterioriliac crest into lithium-heparin anticoagulant-containing tubes.

BMMNC are prepared by density gradient separation using Lymphoprep™(Nycomed Pharma, Oslo, Norway) as previously described (Zannettino, A.C. et al. (1998) Blood 92: 2613-2628). Following centrifugation at 400×gfor 30 minutes at 4° C., the buffy layer is removed with a transferpipette and washed three times in “HHF”, composed of Hank's balancedsalt solution (HBSS; Life Technologies, Gaithersburg, Md.), containing5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

STRO-3⁺ (or TNAP⁺) cells were subsequently isolated by magneticactivated cell sorting as previously described (Gronthos et al. (2003)Journal of Cell Science 116: 1827-1835; Gronthos, S. and Simmons, P. J.(1995) Blood 85: 929-940). Briefly, approximately 1-3×10⁸ BMMNC areincubated in blocking buffer, consisting of 10% (v/v) normal rabbitserum in UHF for 20 minutes on ice. The cells are incubated with 200 μlof a 10 μg/ml solution of STRO-3 mAb in blocking buffer for 1 hour onice. The cells are subsequently washed twice in IMF by centrifugation at400×g. A 1/50 dilution of goat anti-mouse γ-biotin (SouthernBiotechnology Associates, Birmingham, UK) in HHF buffer is added and thecells incubated for 1 hour on ice. Cells are washed twice in MACS buffer(Ca²⁺- and Mn²⁺-free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01%sodium azide) as above and resuspended in a final volume of 0.9 ml MACSbuffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0.5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb(deposited on 19 Dec. 2005 with American Type Culture Collection (ATCC)under accession number PTA-7282 - see International Publication No. WO2006/108229). After addition of a further 1 ml MACS buffer, the columnis removed from the magnet and the TNAP⁺ cells are isolated by positivepressure. An aliquot of cells from each fraction can be stained withstreptavidin-FITC and the purity assessed by flow cytometry.

Example 2: Cells Selected by STRO-3 mAb are STRO-1^(bright) Cells

Experiments were designed to confirm the potential of using STRO-3 mAbas a single reagent for isolating cells STRO-1^(bright) cells.

Given that STRO-3 (IgG1) is a different isotype to that of STRO-1 (IgM),the ability of STRO-3 to identify clonogenic CFU-F was assessed bytwo-color FACS analysis based on its co-expression with STRO-1⁺ cellsisolated using the MACS procedure (FIG. 1). The dot plot histogramrepresents 5×10⁴ events collected as listmode data. The vertical andhorizontal lines were set to the reactivity levels of <1.0% meanfluorescence obtained with the isotype-matched control antibodies, 1B5(IgG) and 1A6.12 (IgM) treated under the same conditions. The resultsdemonstrate that a minor population of STRO-1^(bright) cellsco-expressed TNAP (upper right quadrant) while the remaining STRO-1⁺cells failed to react with the STRO-3 mAb. Cells isolated by FACS fromall four quadrants were subsequently assayed for the incidence of CFU-F(Table 1).

TABLE 1 Enrichment of human bone marrow cells by dual-color FACSanalysis based on the co-expression of the cell surface markers STRO-1and TNAP (refer to FIG. 1). FACS sorted cells were cultured understandard clonogenic conditions in alpha MEM supplemented with 20% FCS.The data represents the mean number of day 14 colony-forming cells(CFU-F) per 10⁵ cells plated ± SE (n = 3 different bone marrowaspirates). These data suggest that human MPC are exclusively restrictedto the TNAP positive fraction of BM which co-express the STRO-1 antigenbrightly. Frequency of CFU-F/ Enrichment Bone Marrow Fraction 10⁵ Cells(Fold Increase) Unfractionated  11.0 ± 2.2 1.0 BMMNCTNAP⁺/STRO-1^(bright) 4,511 ± 185 410 TNAP⁺/STRO-1^(dull) 0.0 0.0

Example 3: Therapeutic Application of Ovine MPCs in a Sheep Model ofOvine Asthma 3.1 Methods

A house dust mite (HDM) sheep model of allergic asthma was selected forstudying the effect of MPCs on asthma because it uses an allergen thatis clinically relevant to humans. Other models of asthma suffer fromdeficiencies. For example, the mouse OVA challenge model uses anallergen that is not clinically relevant to humans, the pattern anddistribution of pulmonary inflammation is different from those observedin humans, both lung and panchymal inflammation/remodeling are observedand large increases in airway smooth muscle is not observed in contrastto chronic asthma in humans. Similarly, the Ascaris sheep model ofasthma does not make use of a clinically relevant antigen and has astrong neutrophilic response together with a comparatively weakeosinophilic response to the allergen (Ascaris saum) not normallyexposed in humans.

Asthma was initiated in sheep by administration of three subcutaneousinjections of house dust mite antigen (50 μg) with alum two weeks apart.Sheep showing high IgE responses as detected by ELISA were thenselected, with a 1.5-fold increase in IgE levels following antigenadministration considered a “high IgE response”.

On days 7, 28 and 49 sheep received aerosol challenges with house dustmite antigen (5 mL containing 200 μg/mL antigen) using a nebulizerconnected to a mechanical ventilator. The mechanical ventilator assiststhe sheep to breathe for 10 minutes at 20 breaths per minute so thateach sheep received a dose of 200 breaths of aerosolized antigen perchallenge. This challenge has been previously shown to be sufficient toinduce asthmatic and inflammatory responses in sheep.

On days 7, 28 and 49 bronchial hyperresponsiveness was quantitated bycalculating the dose-response to increasing concentrations of thebrochoconstrictor carbachol. The expected dose range for this test isbetween 5-300 breaths of 1 mg/ml aerosilized carbachol to give a 100%increase in resistance.

Early phase asthmatic responses were also measured , with the expectedrange of responses being between 50% and 900% change in resistance afterantigen administration compared with baseline (pre-antigenadministration) values.

Sheep were then randomized into four groups as shown in Table 2, suchthat all groups contain sheep with a similar range of physiologicalresponses. Ovine MPCs (passage 5) in ProFreeze™/DMSO/αMEM were dilutedin saline and then administered to the relevant groups on day 63 (twoweeks after the administration of the third aerosol challenge ofantigen). A summary of the treatment protocol is shown in FIG. 3.

TABLE 2 Treatment groups Treatment Group No of animals Type Dose A 10House dust mite 25 million MPCs allergen and MPCs B 11 House dust mite75 million MPCs allergen and MPCs C 10 House dust mite 150 million MPCsallergen and MPCs D 11 House dust mite N/A allergen and saline (control)

Ovine MPCs were administered by intravenous infusion (100 mL/30 minutes)into the jugular vein. Sheep were then challenged again one and fourweeks (days 70 and 91, respectively) with house dust mite allergen.

The measurement of baseline lung function (early asthmatic response[EAR]) was performed by assessing esophageal and tracheal pressures andpulmonary airflows to calculate airway resistance, as previouslydescribed by Koumoundouros et al., Exp. Lung Res., 32: 321-330, 2006. Inthis protocol, a balloon catheter was inserted nasally into the loweresophagus to measure esophageal pressure (i.e. external pressure). Tomeasure internal airway pressure, a tracheal catheter was placed in anasally inserted endotracheal tube. Airflow was measured via apneumotachograph (Hans Rudolph, Kansas City, USA) attached to theproximal end of the endotracheal tube. The esophageal and trachealcatheters, and pneumotachograph were connected to differentialtransducers which allow the measurement of transpulmonary pressuretogether with airflow. Digital data from these recordings were analyzedin a customized Labview Pty Ltd software program, to record airwayresistance on a breath-by-breath basis. Allergen-inducedbronchoconstriction was measured in sheep by analyzing airway resistancechanges at specific times after an aerosol challenge with HDM.Resistance values were recorded for one hour immediately after thischallenge, to assess the early phase asthmatic response (EAR), thenrerecorded 6 hours after challenge to assess the 6 hour asthmaticresponses. The results for EAR are expressed as the percentage change inairway resistance from baseline resistance values after an aerosolizedsaline challenge to peak resistance values over the first hour after HDMchallenge. The LAR data are expressed as the percentage change in airwayresistance from baseline resistance values to average resistance valuesmeasured six hour after HDM challenge.

Bronchial hyperresponsiveness (BHR), also called airwayhyperresponsiveness (AHR), is a measure of the reactivity of airwayclosure in response to a non-specific stimulant. Asthmatic airways arenotoriously twitchy, and react to relatively low doses ofbronchoconstricting agents such as the cholinergic agonists carbacholand methacholine. In sheep, BHR was assessed before the start of the HDMchallenge period, and then after several weeks of HDM challenges. Thiswas achieved by administering the bronchoconstrictor carbachol in arange of doubling aerosol doses (0.25%-4% w/v carbachol) and measuringchanges in airway resistance immediately after each dose of carbachol.The results were expressed as the concentration of carbachol aerosolneeded to increase airway resistance by 100% from baseline or themaximum dose of carbicol has been reached. The concentration of theadministered carbachol dose was measured in Breath Units (BUs); one BUis one breath of 1% w/v carbachol. Sheep that have been sensitized inthe lungs to HDM usually bronchoconstrict with relatively low doses ofcarbachol.

Approximately 20-30 mL of blood was collected on Day -7 (study entrybaseline) and 24 h after BHR testing (Days 2, 51, 72, 93) and thefollowing assays performed:

-   Hematology and Coagulation: red blood cell count (RBC), white blood    cell count (WBC), hemoglobin (Hb), hematocrit, platelets,    neutrophils, lymphocytes, monocytes, eosinophils, basophils,    fibrinogen.-   Biochemistry: sodium, potassium, chloride, bicarbonate, glucose,    creatinine, calcium, magnesium, phosphate, total protein, albumin,    total bilirubin, aspartate transaminase (AST), alanine transaminase    (ALT), gamma-glutamyl transpeptidase (GGT).-   Cytokine testing: Cytokines (TNF-α and IFN-γ)

Serum samples collected at Day 49, Day 63, and Day 91 were assayed forthe presence of IgE by standard enzyme-linked immunosorbent assay(ELISA).

Broncho-alveolar lavage (BAL) cells were collected by infusing 10 mLsaline into the left lung using a fiber-optic bronchoscope andrecovering BAL cells and fluid. BAL cells were differentially stainedwith Haem Kwik (HD Scientific Pty Ltd.) stain to ascertain thepercentages of the various leukocytes present. BAL was performed onDay-7 (study entry baseline) and 24 h after BHR testing (Days 2, 51, 72,93).

Animals were euthanized using an overdose of pentobarbitone sodium (atleast 200 mg/kg; i.e. 20 mL of a 400 mg/mL solution per 40 kg sheep.

Necropsy and tissue collection are performed on all animals that die orare euthanized.

Tissue samples were collected at necropsy (Day 93) from the left caudallung field, and frozen in OCT embedding medium in molds on aluminiumtrays floating on liquid nitrogen for immunohistochemistry. Two tissueblocks were frozen per sheep lung. Frozen sections (5 μm) were stainedusing mAbs against the sheep cell surface molecules CD4, CD8, CD45R, γδ,and IgE. Eosinophils were identified after tissue staining withendogenous peroxidase and counterstained with hematoxylin and eosin-y.Individual cells were examined and counted in the parenchyma, airwaylamina propria, and outer airway wall for each sheep, and expressed asthe number of cells per mm² of tissue examined. For high density celltypes, at least one hundred cells were counted in the respective areas,at 200 times magnification, to ascertain the cell density. For lowdensity cell types, the density was calculated from nonoverlappingfields taken from the all the relevant areas from the complete section.All cell identification, counting, and density calculations wereperformed by observers who were blinded to the treatment groups.

3.2 Results Early Phase Asthmatic Response (EAR) in HDM-SensitizedAsthmatic Sheep at Pre-Treatment, 1 and 4 weeks After a SingleIntravenous Infusion of oMPC or Saline

Sheep which received 150 million oMPCs had significantly improved lungfunction during the hour after allergen challenge at the 4 week postoMPC treatment time point (FIG. 4A, B & C). The improvement in lungfunction at the 4 week post oMPC treatment time point manifested as a57.1% reduction in EAR after allergen challenge when compared topre-oMPC treatment EAR (p<0.05 (FIG. 4A & C).

Late Phase Asthmatic Response (LAR) in HDM-Sensitized Asthmatic Sheep atPre-treatment, 1 and 4 Weeks after a Single Intravenous Infusion of oMPCor Saline

The saline control group showed a trend toward increase in LAR at sixhours after allergen challenge when assessed at both the 1 week and 4week time points when compared to pretreatment values (FIG. 5A). The 25million oMPC treatment group was associated with a significant declinein 6 hour LAR at the 1 week when compared to pretreatment values (FIG.5A). The 75 and 150 million oMPC treatment groups all experienced atrend towards a decline in 6 hour LAR at both the 1 and 4 weekpost-treatment time points when compared to pretreatment values. Asummary graph showing the comparative changes between the treatmentgroups in the 6 hour LAR is shown in FIG. 5B. When evaluating therelative change in LAR by a percent change from pre-treatment tofollow-up treatment, the percentage change in LAR in the 25 million oMPCdose group was significantly improved compared to control at the 1 weektime point (p<0.05, FIG. 5B). Similar trends were shown for the 6 hourLAR at the 4 week post oMPC treatment time point (FIG. 5C).

Bronchial Hyperresponsiveness (BHR) in Asthmatic Sheep at Pre-Treatment,1 and 4 Weeks after a Single Intravenous Infusion of oMPC or Saline

The control group which received saline vehicle treatment in lieu ofoMPCs experienced no significant changes in BHR at the 1 week and 4 weektime points (FIG. 6A, B, C & D). The sheep group which received 75million oMPCs had significantly improved MIR indices at both the 1 and 4week time points post oMPC treatment when compared with BHR measuredbefore oMPC treatment (FIG. 6A). The differences between the pretreatment and 1 and 4 week time points were statistically significantwhen all treatment groups were pooled together in a post hoc analysis(FIG. 6D).

Bronchoalveolar Lavage (BAL) Fluid Analysis: Inflammatory Cell Profilein BAL Fluid in Asthmatic Sheep at Pre-Rreatment, 1 and 4 Weeks after aSingle Intravenous Infusion of oMPC or Saline

Bronchoalveolar lavage (BAL) was sampled two days after allergenchallenge at 1 week and 4 weeks after either oMPC or saline-controltreatment. In all sheep used in this trial the mean baseline percentageof eosinophils of total BAL cells sampled before allergen challenge andstem cell treatment is 4.5%. The mean percentage of eosinophils in theBAL of all sheep sampled 2 days after allergen challenges and beforestem cell or saline treatments (i.e. mean pretreatment percentage of BALeosinophils) is 15.0%. Analysis of eosinophils in the BAL fluidrecovered from trial sheep 2 days after an allergen challenge and postoMPC treatment revealed that there was a significant difference betweenthe pre-treatment and 1 week time points for sheep infused with 25million oMPCs (FIG. 7A & B). For the 75 million and 150 million oMPCtreated groups, the differences in eosinophil numbers in the BAL fluidbetween pretreatment and the 1 and 4 week time points did not reachstatistical significance. However, the differences between the pre- andpost-treatment BAL eosinophils at both the 1 and 4 week time points werestatistical significant when all three treatment values were pooled in apost hoc analysis (FIG. 7E).

In all sheep used in this trial the mean percentage of neutrophils oftotal BAL cells sampled two days after allergen challenge, and beforestem cell or saline treatments, is a relatively low 0.89%. Thepercentages of neutrophils in the BAL fluid were significantly lower atthe 1 week time point post oMPC, compared to pretreatment values, forthe 25 and 150 million oMPC treated sheep (FIG. 8A). For the 75 millionoMPC group, the percentages of neutrophils in the BAL fluid weresignificantly lower at the 4 week post oMPC time point, compared topretreatment values (FIG. 8A).

Cytospot analysis of lymphocytes and macrophages in the BAL fluidrecovered from all trial sheep 2 days after an allergen challengerevealed that there were no significant differences between the groupsfor any of these cell types in BAL fluid (FIGS. 9-10).

HDM-specific IgE In Sera of Asthmatic Sheep at Pre-Treatment, 1 and 4Weeks after a Single Intravenous Infusion of oMPC or Saline

Since allergen-induced asthma is associated with allergen-specific IgE,the levels for circulating HDM-specific IgE in the sera of all sheepwere assessed before oMPC administration, and at two time points after,oMPC treatments at weeks 1 and 4 (FIG. 11A). The results show that the150 million oMPC dose of was effective in significantly reducingHDM-specific IgE 1 week after oMPC treatment compared with pretreatmentlevels of HDM-specific IgE (FIG. 11A). The 25 and 75 million oMPCtreatments significantly reduced the HDM-specific IgE 4 weeks after oMPCtreatment compared with pretreatment levels of HDM-specific IgE. Thelevels of HDM-specific IgE dropped slightly in the saline-treatedcontrol sheep at the 1 and 4 week time points compared to thepretreatment values; however this difference was not significant (FIG.11A). A comparison between control sheep and sheep infused withdifferent doses of oMPCs which assessed the percentage change in IgElevels in the sera from pre-treatment to land 4 week post treatments isshown in FIGS. 11B & C.

Immunohistology Analysis of Lung Tissue: Inflammatory Cellular Profilein the Lungs of Asthmatic Sheep 4 weeks after a Single IntravenousInfusion of oMPC or Saline

Immunohistochemistry was performed on lung tissues sampled at autopsyfrom the left caudal lobe from the trial sheep. A panel of cell surfaceantibody markers was used on tissue sections to identify CD4, CD8, andγδ-positive T cell subsets, CD45R -positive cells, and IgE-positivecells (identifies mast cells). Eosinophils were identified asperoxidase-positive staining cells. The relative densities of these celltypes were assessed in three separate lung locations comprising: theparenchyma which included non-airway tissue such as alveolar spaces andalveolar walls; the lamina propria of the airway wall which restrictedthe cell density analysis to the airway wall area between the luminalepithelium and the inner boundary of the airway smooth muscle bundle;and the whole airway wall which included density counts between theairway luminal epithelium and the outer adventitia bordering thealveoli.

An analysis of peroxidase-positive eosinophils on lung tissue sectionssampled from asthmatic sheep indicates that the eosinophil density inthe airway wall of sheep treated with 150 million oMPCs wassignificantly lower compared to the eosinophil density in the airwaywall of saline-treated control sheep (p<0.05). Overall, these dataindicate that the 150 million oMPC dose treatment given 4 weeks prior toautopsy was associated with a lower density of peroxidase-positiveeosinophils in allergen exposed airway walls.

Histopathology Analysis: Eosinophil Infiltration in the Lungs ofAsthmatic Sheep 4 Weeks after a Single Intravenous Infusion of oMPC orSaline and 24 Hours Following Re-Challenge with HDM

Lung tissues were stained with the Luna histological method whichidentifies eosinophils by staining the eosinophil granules a distinctivered colour, with the background tissue staining blue.

Analysis of the findings of bronchiolar luminal debris/eosinophils andatelectasis with eosinophils in the cranial and caudal lungs did notreveal any significant differences between the control and treatedanimals. Analysis of the findings in the cranial and caudal lung lobesof control and treated sheep revealed a trend towards a decrease inLuna-positive eosinophils in animals in Group C (i.e. sheep treated with150 million oMPCs). In the cranial left lung lobe, the incidence of thenumber of sheep showing Luna-positive eosinophils decreased from 5 inthe control group to 3 sheep in the 150 million oMPC group. In thecranial right lobe, the incidence of the number of sheep showingLuna-positive eosinophils decreased from 5 sheep in the control group to4 in the 150 million oMPC treated group. In the caudal left lung lobe,the incidence in the number of sheep with Luna-positive eosinophilsdecreased from 5 sheep in the control group to 3 in the 150 million oMPCtreated group. In the caudal right lung lobe, the incidence in thenumber of sheep with Luna-positive eosinophils decreased from 4 in thecontrol group to 2 in the 150 million oMPC treated-group. There were nodifferences in the incidence of sheep showing Luna-positive eosinophilsbetween the 25 million and 75 million oMPC dose groups and the controlgroup.

Post-hoc analyses were performed to assess whether the 150 million oMPCdose was more effective overall in reducing the presence ofLuna-positive eosinophils compared to the control sheep. This analysiswas performed by adding the number of sheep showing remarkableLuna-positive eosinophil staining for each of the four lung lobesexamined. This analysis showed that while there were lower numbers ofsheep showing Luna-positive eosinophil pathology in the 150 million oMPCgroup compared with the saline-treated control group.

3.3 Discussion

The present study evaluated the safety and efficacy of oMPC therapy inan ovine model of asthma. Sheep with high levels of HDM-specific IgFantibodies in their sera, compared to pre-immunization levels, weregiven three whole lung aerosol challenges with HDM over a 6-week periodto sensitize their airways to HDM. The sheep were randomly allocatedinto four groups and given either saline (control), or one of threedoses of oMPC treatments (25, 75, or 150 million oMPCs) by IV infusion.The sheep were then re-challenged with HDM at 7 and 28 days after theirrespective oMPC or saline treatments. Lung function and BAL cellanalyses were assessed soon after HDM re-challenge at the time pointspreviously indicated. IV infusion of oMPCs at 25, 75, or 150 millionoMPCs was well tolerated and was without adverse events that wereassociated with the administration of these cells.

In the current study, the i.v. infusion of a single dose of oMPCs isassociated with generally less severe physiological responses toallergen challenges compared to control sheep. For example, there wasstatistically significant attenuation of EAR lung function responses atfour weeks after treatment with 150 million oMPCs. Interestingly, thesignificant reduction on EAR was delayed and observed at 4 weekspost-MPC treatment in the 150 million oMPC treatment group. Withoutbeing bound by any theory or mode of action, this delayed effect ispotentially due to the long half-life of membrane bound mast cellallergen-specific IgE. This may indicate that after allergen-specificIgF is eventually shed from mast cells, MPCs are then able to reducemast cell degranulation which ultimately results in a reduction in EARat 4 weeks after oMPC treatment.

All three oMPC treatment groups generally have attenuated LAR lungfunction indices at 1 week post oMPC treatment compared to control. Ananalysis of pooled data from the three different doses of oMPCs showsthat oMPC treatments statistically significantly improved BHR lungfunction indices compared to control saline-infused sheep at both the 1week and 4 week post-treatment time points. Thus, the improvement in theBHR lung function indices for oMPC-treated sheep appeared to persist for4 weeks after oMPC infusion. This is consistent with the interpretationof the EAR data from the 150 million dose group which indicates thatsignificant treatment effects of oMPC infusion are apparent at the 4week time point.

Without being bound by any theory or mode of action, the effects ofoMPCs in improving lung function in sheep with experimental asthma maybe related, at least in part, to the somewhat lower density ofeosinophils in the airway wall in these oMPC-treated animals. Theblinded morphometric analyses of the tissue densities of eosinophils inthe airway wall showed that the 150 million oMPC treated group had asignificantly lower density of tissue eosinophils compared with thesaline-treated control sheep. Moreover, the highest oMPC dose waseffective in reducing airway eosinophil density over the 4 week studyperiod, given that all the morphometric data for the analyses werecollected at autopsy four weeks after a single dose of oMPCs.

The results show that oMPC treatments are associated with lower levelsof neutrophils in the BAL fluid. The mean percentage of neutrophils intotal BAL cells two days after allergen challenges in all trial sheepbefore treatment is 0.89%. From this relatively low percentage, the 25and 150 million oMPC treatments effectively reduced the percentneutrophils in the BAL by over 50% at the 1 week time point post oMPCs.At the later 4 week time point after oMPC administration, treatment with75 million oMPCs significantly reduced the neutrophils in the BAL. Thepresence of neutrophils in the BAL fluid and airway walls has beenassociated with the pathology of certain phenotypes of asthma.

All sheep used in the study were selected into trial on the basis ofhigh levels of HDM-specific IgE antibodies in their sera seven daysafter the completion of peripheral immunizations with HDM, and thereforeonly sensitized sheep were used in the study. It is noteworthy that theresult shows that oMPC treatment attenuates allergen-specific IgEantibodies and that the effects last for four weeks after a singleinfusion of either 25 or 75 million oMPCs. In the 150 million oMPCgroup, the dampening of IgF was significant at 1 week post oMPC and wasreduced at the four week sampling time point. Saline-treated controlsheep did not show significant reductions in the levels of serumHDM-specific IgE at either the 1 or 4 week time points compared withpre-treatment values.

1. A method of treating or preventing a respiratory condition and/or fortreating an IgE-mediated allergy and/or for reducing an allergicresponse to an allergen and/or for inducing anergy to an allergen in asubject and/or improving lung function in a subject suffering from anallergy, the method comprising administering to the subject a populationof cells enriched for STRO-1⁺ cells and/or progeny thereof and/orsoluble factors derived therefrom.
 2. The method of claim 1, wherein therespiratory condition is an acute respiratory condition or a chronicrespiratory condition.
 3. The method of claim 1 or claim 2, wherein therespiratory condition is an inflammatory respiratory condition, anobstructive respiratory condition or a restrictive respiratorycondition.
 4. The method of claim 3, wherein the respiratory conditionor allergy is an obstructive respiratory condition or allergy or aninflammatory lung condition or allergy.
 5. The method of claim 4,wherein the respiratory condition is asthma.
 6. The method of claim 5,wherein the asthma is acute asthma, chronic asthma, severe asthma and/orrefractory asthma.
 7. The method of claim 6, wherein the asthma is longacting beta agonist (LABA) refractory asthma or steroid refractoryasthma.
 8. The method of claim 3, wherein the respiratory condition is arestrictive respiratory condition.
 9. The method of claim 8, wherein therespiratory condition is idiopathic pulmonary fibrosis.
 10. The methodof claim 1, wherein the condition is allergy to house dust mite allergen(HDM) or the allergen is HDM.
 11. The method of any one of claims 1 to10 comprising administering a population of cells enriched forSTRO-1^(bright) cells and/or progeny thereof and/or soluble factorsderived therefrom.
 12. The method of any one of claims 1 to 11, whereinthe population enriched for STRO-1⁺ cells and/or progeny thereof and/orsoluble factors derived therefrom are administered systemically.
 13. Themethod of claim 12, wherein the population enriched for STRO-1⁺ cellsand/or progeny thereof and/or soluble factors derived therefrom areadministered intravenously or intranasally.
 14. The method of any one ofclaims 1 to 13, wherein the population and/or the progeny and/or thesoluble factors are administered a plurality of times.
 15. The method ofany one of claims 1 to 14, comprising the subject and administering afurther dose of the population and/or the progeny and/or the solublefactors when one or more of the following occurs: (i) a subject beginsto persistently wheeze and/or cough and/or have chest tightness and/orhave difficulty breathing; (ii) a subject shows one or more of thefollowing when assessed by spirometer: a) 20% difference on at leastthree days in a week for at least two weeks; b) ≥20% improvement of peakflow following treatment with: 10 minutes of inhaled β-agonist; sixweeks of inhaled corticosteroid; 14 days of 30 mg prednisolone. c) ≥20%decrease in peak flow following exposure to a trigger; (iii)bronchoscopy showing abnormal cells and/or foreign substances and/orblockages in the respiratory tract of a subject; or (iv) chest CT scanshowing abnormalities of the blood vessels in the lungs, accumulation ofblood or fluid in the lungs, bronchiectasis, pleural effusion orpneumonia.
 16. The method of any one of claims 1 to 15 comprisingadministering a dose of the population and/or the progeny and/or thesoluble factors sufficient to achieve one or more of the following: (i)improved bronchial hyperresponsiveness; (ii) reduced eosinophilinfiltration of the lung or bronchoalveolar lavage fluid; (iii) reducedneutrophil infiltration of the lung or bronchoalveolar lavage fluid;(iv) reduced late asthmatic response; (v) reduced early asthmaticresponse; and/or (vi) reduced lung remodeling/fibrosis.
 17. The methodof any one of claims 1 to 16 comprising administering between 1×10⁶ to150×10⁶ STRO-1⁺ cells and/or progeny thereof.
 18. The method of any oneof claims 1 to 17 comprising administering a whole body dose of STRO-1⁺cells and/or progeny thereof and/or soluble factors derived therefrom.19. The method of claim 18 comprising administering 150×10⁶ STRO-1⁺cells and/or progeny thereof in 10 mL to the subject.
 20. The method ofany one of claims 1 to 19, wherein the population enriched for STRO-1⁺cells and/or progeny cells are autogeneic or allogeneic and/or thesoluble factors are derived from autogeneic or allogeneic cells.
 21. Themethod of any one of claims 1 to 20, wherein the population enriched forSTRO-1⁺ cells and/or progeny cells have been culture expanded prior toadministration and/or prior to obtaining the soluble factors.
 22. Themethod of any one of claims 1 to 21, wherein the population enriched forSTRO-1⁺ cells are STRO-1^(bri), and/or express tissue non-specificalkaline phosphatase (TNAP) and/or the progeny cells and/or solublefactors are derived from STRO-1⁺ cells that are STRO-1^(bri) and/orexpress TNAP.
 23. The method according to any one of claims 1 to 22,wherein the STRO-1⁺ cells and/or progeny cells thereof and/or solublefactors derived therefrom are administered in the form of a compositioncomprising said STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom and a carrier and/or excipient.
 24. Apopulation of cells enriched for STRO-1⁺ cells and/or progeny thereofand/or soluble factors derived therefrom for use in the treatment orprevention of a respiratory condition and/or for treating anIgE-mediated allergy and/or for reducing an allergic response to anallergen and/or for inducing anergy to an allergen and/or improving lungfunction in a subject suffering from an allergy.
 25. Use of a populationof cells enriched for STRO-1⁺ cells and/or progeny thereof and/orsoluble factors derived therefrom in the manufacture of a medicament fortreating or preventing a respiratory condition in a subject and/or fortreating an IgE-mediated allergy and/or for reducing an allergicresponse to an allergen and/or for inducing anergy to an allergen and/orimproving lung function in a subject suffering from an allergy.